Reaction mass of Pentaerythritol bis (2-ethylhexanoate) bis (3,5,5-trimethylhexanoate) and Pentaerythritol tris (2-ethylhexanoate) 3,5,5-trimethylhexanoate and Pentaerythritol 2-ethylhexanoate tris (3,5,5-trimethylhexanoate) and Pentaeryhthritol tetrakis(2-ethylhexanoate) and Pentaerythritol tetrakis (3,5,5-trimethylhexanoate)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A Reproduction/Developmental Toxicity Screening Test performed according to OECD guideline 421 and GLP principles is available. In this study, RB68 was dosed via the oral route at 100, 300 or 1000 mg/kg bw/day, no adverse effects were seen up to and including the highest dose level tested. As a consequence, the parental, reproduction and developmental NOAELs were found to be at least 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 2019 - 22 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-12 weeks (males); 12-14 weeks (females)
- Weight at study initiation: 291-329 g (males); 200-236 g (females)
- Fasting period before study: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
- Housing: During pre-mating period: group housing (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm); During mating: males and females cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm); During post-mating phase: males housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage, females individually housed in Macrolon plastic cages (MIII type, height 18 cm); During lactation: Macrolon plastic cages (MIII type, height 18 cm), pups were housed with the dam. The cages contained appropriate bedding and were equipped with water bottles. For psychological/environmental enrichment and nesting material, animals were provided with paper.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum (except males o/n before sacrifice)
- Water: Municipal tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS (ACTUAL)
- Temperature (°C): 19-22
- Humidity (%): 52-68
- Air changes (per hr): 10 or more (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES from 27 August 2019 to 31 October 2019

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Stability is confirmed for at least 24 hours at room temperature under normal laboratory light conditions for both levels, at least 8 days in the refrigerator for both levels and at least 3 weeks in the freezer (≤ -15°C) for the high level (low level not stable) over the concentration range 1 to 200 mg/mL (solutions) (Charles River Project 20195988).

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females had not shown evidence of mating were separated from their males. Since less than 9 females per group had shown evidence of mating, each non-mated female was re-mated once with a male of proven fertility of the same group for 1-9 days. Detection of mating was not confirmed in first instance for one female from the mid dose group. Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The accuracy of preparation and homogeneity of RB68 in the formulations was determined using the analytical method validated for the test item in vehicle in Charles River project 20195988. Due to problems with the stability of the analytical system during analysis of Week 1 formulations, no results were obtained. The QC and formulation samples were re-analyzed together with fresh QC samples prepared at concentration levels corresponding with the concentration levels from the formulation samples. The mean accuracies of the first QC samples were not within the criterion range of 90-110%. Also, not all the results of the freshly prepared QC samples analysed in the same series did meet the criteria. Therefore, it was decided to correct the study samples using the mean recoveries of the fresh QC samples prepared and to perform additional analysis of formulations prepared for use in Week 3.

Duration of treatment / exposure:

Males were treated for 29-38 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-55 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 27-29 days.

Frequency of treatment:

Once daily (7 days/week)

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

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

Remarks:

Mid dose group

Dose / conc.:

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

Remarks:

High dose group

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 the results of a 10-day Dose Range Finder (DRF) with oral administration of RB68 in rats and in an attempt to produce graded responses to the test item.
The procedures in the DRF were identical to the procedures in the main study, except for the following: no dose formulation analyses, 3 females/ dose group, administration for 10 consecutive days, terminal body weight, kidney and liver weight were determined, no histopathology performed.

Positive control:

No.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing) , and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no toxicologically relevant changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded.

Blood was collected on the day of scheduled necropsy for thyroid hormone analysis. Measurement of total T4 was conducted for F0-males. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no toxicologically relevant changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded.

Oestrous cyclicity (parental animals):

Oestrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.

Sperm parameters (parental animals):

For the testes of all males of the control and the high dose group, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or anogenital distance, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
Mortality/ moribyundity: Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter.
Clinical observations: At least once daily.
Body weights: Live pups were weighed individually on PND 1, 4, 7 and 13.
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance was measured for all live pups on PND 1. The anogenital distance was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.
Blood of F1-animals was collected on PND 4 and PND 14-16. Measurement of total T4 was conducted for PND 14-16 pups. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no adverse changes in T4 were noted in pups at PND 14-16.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after a minimum of 29-38 days of administration
- Maternal animals: All surviving animals on PND 14-16

GROSS NECROPSY
- Gross necropsy performed

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 14-16 days of age.
Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director.

Measurement of total T4 was conducted for PND 14-16 pups. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no adverse changes in T4 were noted in pups at PND 14-16.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: Low dose group vs. Control, Mid dose group vs. Controls, High dose group vs. Controls. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Reproductive indices:

Parental variables:
Body Weight Gains: Calculated against the body weight on Day 1 (pre-mating, mating and lactation periods) or Day 0 (post-coitum period);
Relative Food Consumption: Calculated against the body weight for scheduled intervals;
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

Reproduction Variables:
Mating index (%): (Number of females mated/ Number of females paired;
Precoital time: Number of days between initiation of cohabitation and confirmation of mating;
Fertility index (%): (Number of pregnant females/ Number of females mated) x 100;
Gestation index (%): (Number of females with living pups on Day 1/ Number of pregnant females) x 100;
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition.

Offspring viability indices:

Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100;
Live birth index (%): (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100;
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/ Number of live pups at First Litter Check) x 100;
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100;
Viability index (%): (Number of live offspring on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100;
Lactation index (%): (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment. One female of the control group and one female at 300 mg/kg bw/day died during blood sampling on the day of scheduled necropsy. These mortalities were considered procedure-related and not caused by the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effects on body weight and body weight gain were noted in all groups of treated males, and in females treated up to 300 mg/kg bw/day. In females treated at 1000 mg/kg bw/day, mean body weight gain was slightly decreased during the post-coitum period, reaching statistical significance from Day 4 to 11 post-coitum. Since values remained within the historical control range, these changes were considered non-adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight was similar to the control level over the treatment period.

Description (incidence and severity):

No toxicologically relevant changes were noted in serum T4 levels in F0-males. At 300 mg/kg bw/day, total T4 levels in F0-males were increased (1.18x of control). Values were just outside the historical control range. In the absence of a dose-related trend, this was considered not toxicologically relevant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment with RB68 were noted in the kidneys of males, a table with the exact results is incuded below. Hyaline droplet accumulation in the kidney was present in all treated males at an increased incidence and severity (up to moderate). Tubular basophilia in the kidney was observed at increased incidence and severity in males treated at 1000 mg/kg bw/day (up to slight). The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females had regular cycles of 4 days.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were 1/10 couples treated at 300 mg/kg bw/day and 2/10 at 1000 mg/kg/day not pregnant. Evidence of mating was observed within 4 days in 10/10 females for the control and 100 mg/kg bw/day groups, 9/10 females for the 300 mg/kg bw/day group and 8/10 females for the 1000 mg/kg bw/day group. One female at 1000 mg/kg bw/day showed evidence of mating within 13 days. One female at 300 and one female at 1000 mg/kg bw/day mated within 1-2 days during the second pairing period, with a different male. One male at 300 mg/kg bw/day and one male at 1000 mg/kg bw/day did not mate. Histopathology did not reveal any changes in the reproductive organs, and this incidence was considered to be within the normal range. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

Precoital time was considered not to be affected by treatment with the test item. Number of implantation sites was considered not to be affected by treatment with the test item.
The fertility indices were 100% for the control and 100 mg/kg/day groups, and 90% and 80% for the 300 and 1000 mg/kg/day groups, respectively. Fertility index was considered not to be affected by treatment with the test item.
No signs of difficult or prolonged parturition were noted among the pregnant females.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen up to and including 1000 mg/kg bw/day

Remarks on result:

other: Effects on male kidneys (increased weight at 1000 mg/kg bw/day and histopathological changes in all dosed males (hyaline droplet accumulation) were considered rat-specific and not relevant for humans

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment with the test item.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum T4 levels in male PND 14-16 pups were considered not to be affected by treatment with the test item. Serum T4 levels in female PND 14-16 pups were statistically significantly increased compared with concurrent controls (1.26x of control). This change was considered possibly test item-related, but as values remained within the historical control range differences were considered non adverse.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was found not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg bw/day/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test item.
The gestation indices were 100% for all groups.
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 93% for the control and 100 mg/kg bw/day groups, and 96 and 90% for the 300 and 1000 mg/kg bw/day groups, respectively.
Live litter sizes were 11.7, 13.2, 12.0 and 11.4 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
The live birth indices were 99, 100, 99 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Viability index was 98% for all groups. The lactation indices were 99, 96, 100 and 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including 1000 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In a Reproduction/Developmental Toxicity Screening Test according to OECD guideline 421 and GLP principles with exposure to RB68 via the oral route, no adverse effects were seen up to and including the highest dose level tested. As a consequence, the parental, reproduction and developmental NOAELs were found to be at least 1000 mg/kg bw/day.

Executive summary:

A Reproduction/Developmental Toxicity Screening Test was performed according to OECD guideline 421 and GLP principles. Rats were exposed via the oral route to RB68 at dose levels 0, 100, 300 and 1000 mg/kg bw/day. Males were treated for 29-38 days, including a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-65 days, and at least 13 days after delivery. Females which failed to deliver were treated for 46 days. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces. Pups were sacrificed post-natal day 12-14. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously.

No mortality occurred during the study (one control female and one female dosed at 300 mg/kg bw/day died during blood withdrawal on the day of necropsy). No signs of toxicity were noted in the animals. In females treated at 1000 mg/kg bw/day, mean body weight gain was slightly decreased during the post-coitum period, reaching statistical significance from Day 4 to 11 post-coitum. Since values remained within the historical control range, these changes were considered non-adverse. In males, test item-related higher kidney weights were noted at 1000 mg/kg bw/day. Hyaline droplet accumulation was present in all treated males, and tubular basophilia in males at 1000 mg/kg bw/day only. This was considered non-adverse based on the mild degree of the basophilia, and lack of any structural change to the renal architecture. Furthermore, this effect in male kidneys is considered rat specific and not relevant for humans.

There were 1/10 couples treated at 300 mg/kg bw/day and 2/10 at 1000 mg/kg/day not pregnant. One female at 300 and one female at 1000 mg/kg bw/day were not mated. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. Overall it was concluded that no effects were seen on reproduction. Furthermore, no developmental effects were seen in pups of any dose group.

As no adverse effects were seen up to and including the highest dose level tested, the parental, reproduction and developmental NOAELs were concluded to be at least 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:

subacute

Species:

rat

Quality of whole database:

The available study is reliable (Klimisch 1 study).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

A Reproduction/Developmental Toxicity Screening Test performed according to OECD guideline 421 and GLP principles is available. In this study, RB68 was dosed via the oral route at 100, 300 or 1000 mg/kg bw/day, no adverse effects were seen up to and including the highest dose level tested. As a consequence, the parental, reproduction and developmental NOAELs were found to be at least 1000 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 2019 - 22 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-12 weeks (males); 12-14 weeks (females)
- Weight at study initiation: 291-329 g (males); 200-236 g (females)
- Fasting period before study: no
- Housing: During pre-mating period: group housing (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm); During mating: males and females cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm); During post-mating phase: males housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage, females individually housed in Macrolon plastic cages (MIII type, height 18 cm); During lactation: Macrolon plastic cages (MIII type, height 18 cm), pups were housed with the dam. The cages contained appropriate bedding and were equipped with waterbottles. For psychological/environmental enrichment and nesting material, animals were provided with paper.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum (except males o/n before sacrifice)
- Water: Municipal tap water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS (ACTUAL)
- Temperature (°C): 19-22
- Humidity (%): 52-68
- Air changes (per hr): 10 or more (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES from 27 August 2019 to 31 October 2019

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Stability is confirmed for at least 24 hours at room temperature under normal laboratory light conditions for both levels, at least 8 days in the refrigerator for both levels and at least 3 weeks in the freezer (≤ -15°C) for the high level (low level not stable) over the concentration range 1 to 200 mg/mL (solutions) (Charles River Project 20195988).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The accuracy of preparation and homogeneity of RB68 in the formulations was determined using the analytical method validated for the test item in vehicle in Charles River project 20195988. Due to problems with the stability of the analytical system during analysis of Week 1 formulations, no results were obtained. The QC and formulation samples were re-analyzed together with fresh QC samples prepared at concentration levels corresponding with the concentration levels from the formulation samples. The mean accuracies of the first QC samples were not within the criterion range of 90-110%. Also, not all the results of the freshly prepared QC samples analysed in the same series did meet the criteria. Therefore, it was decided to correct the study samples using the mean recoveries of the fresh QC samples prepared and to perform additional analysis of formulations prepared for use in Week 3.

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females had not shown evidence of mating were separated from their males. Since less than 9 females per group had shown evidence of mating, each non-mated female was re-mated once with a male of proven fertility of the same group for 1-9 days. Detection of mating
was not confirmed in first instance for one female from the mid dose group. Evidence of mating was obtained by palpation and indirectly by delivery of a litter. Apparently, mating was overlooked in the assessment of the vaginal lavage, which explains the continued di-estrous during the mating in this female. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.

Duration of treatment / exposure:

Females that delivered were treated for 50-55 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 27-29 days.

Frequency of treatment:

Once daily (7 days/week)

Dose / conc.:

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

Remarks:

Low dose group

Dose / conc.:

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

Remarks:

Mid dose group

Dose / conc.:

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

Remarks:

High dose group

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 the results of a 10-day Dose Range Finder (DRF) with oral administration of RB68 in rats and in an attempt to produce graded responses to the test item.
The procedures in the DRF were identical to the procedures in the main study, except for the following: no dose formulation analyses, 3 females/ dose group, administration for 10 consecutive days, terminal body weight, kidney and liver weight were determined, no histopathology performed.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing) , and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was quantitatively measured weekly, except for females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no toxicologically relevant changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded.

Oestrous cyclicity (maternal animals)
Oestrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included the number of (former) implantations. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Fetal examinations:

Mortality/ moribundity: Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter.
Clinical observations: At least once daily.
Body weights: Live pups were weighed individually on PND 1, 4, 7 and 13.
Sex was externally determined for all pups on PND 1 and 4.
Anogenital distance was measured for all live pups on PND 1. The anogenital distance was normalized to the cube root of body weight.
All male pups in each litter were examined for the number of areola/nipples on PND 13.
Blood of F1-animals was collected on PND 4 and PND 14-16. Measurement of total T4 was conducted for PND 14-16 pups. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no adverse changes in T4 were noted in pups at PND 14-16.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.

Postmortem examinations (offspring)
SACRIFICE
- The F1 offspring were sacrificed at 14-16 days of age. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: Low dose group vs. Control, Mid dose group vs. Controls, High dose group vs. Controls. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test ). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Indices:

Maternal variables:
Body Weight Gains: Calculated against the body weight on Day 1 (pre-mating, mating and lactation periods) or Day 0 (post-coitum period);
Relative Food Consumption: Calculated against the body weight for scheduled intervals;
Organ Weight Relative to Body Weight: Calculated against the terminal body weight.

Offspring viability indices:
Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100;
Live birth index (%): (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100;
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/ Number of live pups at First Litter Check) x 100;
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100;
Viability index (%): (Number of live offspring on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100;
Lactation index (%): (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100.

Historical control data:

Included in the report.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment. One female of the control group and one female at 300 mg/kg bw/day died during blood sampling on the day of scheduled necropsy. These mortalities were considered procedure-related and not caused by the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No effects on body weight and body weight gain were noted in females treated up to 300 mg/kg bw/day. In females treated at 1000 mg/kg bw/day, mean body weight gain was slightly decreased during the post-coitum period, reaching statistical significance from Day 4 to 11 post-coitum. Since values remained within the historical control range, these changes were considered non-adverse.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 93% for the control and 100 mg/kg/day groups, and 96 and 90% for the 300 and 1000 mg/kg/day groups, respectively.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Litter size was considered not affected by treatment with the test item. Live litter sizes were 11.7, 13.2, 12.0 and 11.4 living pups/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were considered not to be affected by treatment with the test item. The gestation indices were 100% for all groups.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Fertility index was considered not to be affected by treatment with the test item. The fertility indices were 100% for the control and 100 mg/kg/day groups, and 90% and 80% for the 300 and 1000 mg/kg/day groups, respectively.

Details on maternal toxic effects:

No signs of difficult or prolonged parturition were noted among the pregnant females.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: No adverse effecst observed up to and ncluding the highest dose tested (1000 mg/kg bw/day)

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment with the test item.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the test item. The live birth indices were 99, 100, 99 and 100% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test item.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
Treatment up to 1000 mg/kg/day/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment with the test item. Viability index was 98% for all groups.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item. The lactation indices were 99, 96, 100 and 98% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed up to and including 1000 mg/kg bw/day

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

In a Reproduction/Developmental Toxicity Screening Test according to OECD guideline 421 and GLP principles with exposure to RB68 via the oral route, no adverse effects were seen up to and including the highest dose level tested. As a consequence, the parental, reproduction and development al NOAELs were found to be at least 1000 mg/kg bw/day.

Executive summary:

A Reproduction/Developmental Toxicity Screening Test was performed according to OECD guideline 421 and GLP principles. Rats were exposed via the oral route to RB68 at dose levels 0, 100, 300 and 1000 mg/kg bw/day. Males were treated for 29-38 days, including a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-65 days, and at least 13 days after delivery. Females which failed to deliver were treated for 46 days. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/ feces. Pups were sacrificed post-natal day 12-14. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. No mortality occurred during the study (one control female and one female dosed at 300 mg/kg bw/day died during blood withdrawal on the day of necropsy). No signs of toxicity were noted in the animals. In females treated at 1000 mg/kg bw/day, mean body weight gain was slightly decreased during the postcoitum period, reaching statistical significance from Day 4 to 11 post-coitum. Since values remained within the historical control range, these changes were considered non-adverse. In males, test itemrelated higher kidney weights were noted at 1000 mg/kg bw/day. Hyaline droplet accumulation was present in all treated males, and tubular basophilia in males at 1000 mg/kg bw/day only. This was considered non-adverse based on the mild degree of the basophilia, and lack of any structural change to the renal architecture. Furthermore, this effect in male kidneys is considered rat specific and not relevant for humans. There were 1/10 couples treated at 300 mg/kg bw/day and 2/10 at 1000 mg/kg/day not pregnant. One female at 300 and one female at 1000 mg/kg bw/day were not mated. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. Overall it was concluded that no effects were seen on reproduction. Furthermore, no developmental effects were seen in pups of any dose group.

As no adverse effects were seen up to and including the highest dose level tested, the parental, reproduction and developmental NOAELs were concluded to be at least 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available study is reliable (Klimisch 1 study).

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

A Reproduction/Developmental Toxicity Screening Test was performed according to OECD guideline 421 and GLP principles. Rats were exposed via the oral route to RB68 at dose levels 0, 100, 300 and 1000 mg/kg bw/day. Males were treated for 29-38 days, including a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-65 days, and at least 13 days after delivery. Females which failed to deliver were treated for 46 days. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/ feces. Pups were sacrificed post-natal day 12-14. Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. No mortality occurred during the study (one control female and one female dosed at 300 mg/kg bw/day died during blood withdrawal on the day of necropsy). No signs of toxicity were noted in the animals. In females treated at 1000 mg/kg bw/day, mean body weight gain was slightly decreased during the postcoitum period, reaching statistical significance from Day 4 to 11 post-coitum. Since values remained within the historical control range, these changes were considered non-adverse. In males, test item related higher kidney weights were noted at 1000 mg/kg bw/day. Hyaline droplet accumulation was present in all treated males, and tubular basophilia in males at 1000 mg/kg bw/day only. This was considered non-adverse based on the mild degree of the basophilia, and lack of any structural change to the renal architecture. Furthermore, this effect in male kidneys is considered rat specific and not relevant for humans. There were 1/10 couples treated at 300 mg/kg bw/day and 2/10 at 1000 mg/kg/day not pregnant. One female at 300 and one female at 1000 mg/kg bw/day were not mated. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. Overall it was concluded that no effects were seen on reproduction. Furthermore, no developmental effects were seen in pups of any dose group.

As no adverse effects were seen up to and including the highest dose level tested, the parental, reproduction and developmental NOAELs were concluded to be at least 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on the current data-set, RB68 is not classified and has no obligatory labelling requirement for reproduction and developmental toxicity according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).