3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 May 2020 - 22 Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

version 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

Version 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1‐cyclohexyl‐3‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

EC Number:

604-940-8

Cas Number:

154099-21-5

Molecular formula:

C39 H62 N4 O2

IUPAC Name:

1‐cyclohexyl‐3‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Constituent 2

Reference substance name:

3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

EC Number:

406-370-3

EC Name:

3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

Cas Number:

58890-25-8

Molecular formula:

C27 H36 N4 O2

IUPAC Name:

1-cyclohexyl-3-[4-({4-[(cyclohexylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Constituent 3

Reference substance name:

3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

EC Number:

406-690-3

EC Name:

3,3'-dioctadecyl-1,1'-methylenebis(4,1-phenylene)diurea

Cas Number:

43136-14-7

Molecular formula:

C51 H88 N4 O2

IUPAC Name:

3‐octadecyl‐1‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Thickener (MDI-CHA-ODA)
- Physical description: white powder
- Storage conditions: at room temperature

Specific details on test material used for the study:

The test item is a multi-constituent and considered as 100%. No correction was made for the purity/composition of the test item

Test animals

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: males: 10-11 weeks old, females: 13-14 weeks old
- Weight at study initiation: males: 173 - 315 g; females: 192-244 g
-Fasting period before study: No. Adult males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Females were not fasted overnight.
- Housing: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type) during pregnancy and lactation phase. Pups were housed with the dam.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: municipal tap water ad libitum. Periodic analysis of the water was performed. No known contaminants were present in the feed or water that would interfere with the objectives of the study.
- Acclimation period: 7 days
- Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 24
- Humidity (%): 47 to 71
- Air changes (per hr): Ten or greater with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained

IN-LIFE DATES: From: 10 Jun 2020 To: 17 Aug 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
In order to limit the influence of the absence of formulation analyses, the test item preparation was performed with approved procedures and documented in detail. Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 2 hours after adding the vehicle to the test item. Formulations were prepared protected from light. Dosing formulations containing vehicle only were dosed within a similar timeframe after preparation as test item dosing formulations, when practically possible. Preparations were visually inspected for homogeneity prior to use.

VEHICLE
- Justification for use and choice of vehicle: During the current study, the test item dosing formulations were prepared with corn oil. The main component of corn oil is triglycerides. Corn oil may contain water and/or fatty acids as impurity (making an acidic environment), which may enable hydrolysis of the test substance (Corn oil. Corn Refiners Association, Washington DC, US. 5th Edition, 2006; https://corn.org/wp-content/uploads/2009/12/CornOil.pdf). The test substance Thickener (MDI-CHA-ODA) contains three main components, which have urea groups as only functional group. Urea groups are regarded as non-acids and non-bases and their reactivity as electrophilic species in reactions such as hydrolysis needs extreme conditions (http://webhome.auburn.edu/~deruija/pda1_amides.pdf). Urea groups substituted by aromatic or alkyl groups are stable in acidic and neutral aqueous solutions for at least a year (Chapman, TM. Models for polyurethane hydrolysis under moderately acidic conditions: a comparative study of hydrolysis rates of urethanes, ureas, and amides. J. Polymer Sci: Part A: Polymer Chem. Vol 27, 1993-2005 (1989).). Urea groups do not react with triglycerides, the main component of corn oil. In conclusion, the three main components of Thickener (MDI-CHA-ODA) were expected to be stable in corn oil for the period from formulation till administration.
- Concentration in vehicle: 25, 75, 250 mg/mL
- Amount of vehicle: 4 mL/kg bw
Adjustment was made for specific gravity of the vehicle.

Details on mating procedure:

- M/F ratio per cage: 1:1 mating
- Length of cohabitation: A maximum of 14 days was allowed for mating
- Proof of pregnancy: appearance of intravaginal copulatory plug or by evidence of sperm in vaginal smear, referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility for a maximum of 7 days
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

no

Remarks:

Analysis of test item in vehicle for concentration, homogeneity, and stability was not performed, as no feasible analytical method is available for Thickener (MDI-CHA-ODA) (as demonstrated in Charles River Study No. 20150239).

Duration of treatment / exposure:

Males were treated for 29-37 days, up to and including the day before scheduled necropsy. Females that delivered were treated for 50-68 days. Females which failed to deliver were treated for 42-55 days.

Frequency of treatment:

once daily, 7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

group 2 - low dose

Doses / concentrations 2

Dose / conc.:

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

Remarks:

group 3 - mid dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

group 4 - high dose

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 previously performed 28-day study with oral gavage administration of Thickener (MDI/CHA/ODA) in rats (Charles River Study No. RCC NOTOX 034032). During the 28-day study, dose levels of 0, 50, 200 and 1000 mg/kg bw/day were tested. No treatment-related changes were detected in any of the dose levels and a No Observed Effect Level (NOEL) of 1000 mg/kg bw/day was established. Based on the results of the 28-day study, 100, 300 and 1000 mg/kg bw/day were selected as dose levels. The repeated dose study is summarized in the relevant section.

Positive control:

No, but historical control data are present at Charles River Den Bosch

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least once daily, up to the day prior to necropsy. These clinical observations were conducted after dosing at no specific time.

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

FOOD CONSUMPTION
- 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: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OTHER:
- Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

-Thyroid hormone analysis
Blood of F0-animals was collected on the day of scheduled necropsy at a target volume of 1.0 m. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Blood samples were collected into tubes without anticoagulant, were processed for serum, and serum was analyzed for total Thyroxine (T4).
Measurement of total T4 was conducted for parental males.

Oestrous cyclicity (parental animals):

Estrous 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):

Parameters examined in all adult males: testis weight, epididymis weight.
For the testes of all males in the control and high dose group, and all males that failed to sire, 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
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities: Pups were observed daily for general health/mortality. The number of live and dead pups was determined on PND 1 and daily thereafter.
- Live pups were weighed individually on PND 1, 4, 7 and 13.
- Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Presence of nipples/areolae in male pups: All male pups in each litter were examined for the number of areola/nipples on PND 13.

- Thyroid hormone analysis:
Blood of F1-animals was collected on PND 4 and PND 14-16. On PND 4 at culling, blood was collected from two surplus pups per litter by decapitation and samples were pooled to one sample per litter. On PND 14-16 separate blood samples were collected from two pups per litter. Blood was drawn by aorta puncture under anesthesia. Blood samples were collected into tubes without anticoagulant. were processed for serum, and serum was analyzed for total Thyroxine (T4).
Measurement of total T4 was conducted for PND 14-16 pups.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead, if possible:
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

SACRIFICE
- Parent animals: All animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted. Males (which sired and failed to sire) were euthanized after the completion of the mating period. Females which failed to deliver (with evidence of mating) were euthanized between 25-27 days post-coitum (after confirmation of mating). Females which delivered were euthanized between 14-16 days post partum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera and the appearance of the tissues and organs was observed macroscopically. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. 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.

-Tissue collection and preservation:
Representative samples of the following tissues were collected from all animals and preserved in 10% neutral buffered formalin: cervix, seminal vesicles including coagulation gland, mammary gland, thyroid including parathyroid gland, pituitary gland, prostate gland, ovaries, uterus and vagina. Testes and epididymis were preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours. Gross lesions or masses were also collected from all animals if found.

ORGAN WEIGHTS
At necropsy, for all scheduled euthanasia animals, terminal body weight was measured and the following organs were weighed: epididymis, coagulation gland with seminal vesicle gland, thyroid with parathyroid, prostate and testes. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and
stained with hematoxylin and eosin:
> gross lesions or masses from all animals, if any
> epididymis, thyroid gland, ovaries and testes of all animals of the control and high dose groups
> cervix, epididymis, seminal vesicles including coagulation gland, prostate gland, ovaries, uterus, vagina, testes of all males that fail to sire and females that fail to deliver pups and females
with total litter loss
> mammary gland of females with total litter loss
For the testes of all males of the control and the high dose groups, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Postmortem examinations (offspring):

SACRIFICE
- Pups, younger than 7 days were euthanized by decapitation. On PND 4, the surplus pups were euthanized by decapitation. All remaining pups (PND 14-16) except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
- Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

For all remaining pups euthanized on PND 14-16, 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) were reported when 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:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (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 is significant.
For clinical pathology data Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test. The data corresponding to a response variable of interest and to a related covariate was submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett's test.

Reproductive indices:

Mating index (%): (Number of females mated/Number of females paired) x 100

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female at 1000 mg/kg bw/day presented piloerection on Days 17-18 of treatment and restless behavior, tremors, quick breathing and piloerection between Days 41-43 of treatment. As clinical signs were transient and occurred in a single female only, these were considered incidental and unrelated to treatment with the test item.

Any other clinical signs noted during the treatment period (i.e. alopecia, scars, scabs, wounds) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test item.
Any statistically significant changes in body weight gain (decreased weight gain for the males dosed at 300 mg/kg bw/day in week 2 of dosing) were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Endocrine findings:

no effects observed

Description (incidence and severity):

Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.
An apparent dose-related decrease in mean T4 serum levels was observed in males (mean values were 0.89x, 0.88x and 0.86x of control at 100, 300 and 1000 mg/kg bw/day, respectively). However, no statistical significance was achieved, individual values generally remained within the range of concurrent controls and the concurrent control mean was relatively high mainly due to a relatively high value of one single male. The serum T4 levels were therefore considered unaffected by treatment with the test item (see summary table under section "Attached background material").

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
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.

Two control males, two males at 100 mg/kg bw/day and one male at 1000 mg/kg bw/day had not mated during the 14 days of cohabitation. The females of these couples produced healthy offspring upon subsequent mating with another male of the same dose group (except for one female at 100 mg/kg bw/day that was not pregnant after re-mating). Additionally, one control female, one female at 100 mg/kg bw/day and one female at 300 mg/kg bw/day, were not pregnant despite evidence of mating. No abnormalities were seen in the reproductive organs of these rats, which could account for their lack of offspring (see table under "Any other information on results incl. tables").
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present in all control and high dose males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment with the test item as all females had regular cycles of 4 days.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index was not affected by treatment with the test item. All females showed evidence
of mating, resulting in a mating index of 100% for all groups.
Two males of the control group, two males at 100 mg/kg bw/day and one male at 1000 mg/kg bw/day had not mated during 14 days of cohabitation. The females paired with these males were mated successfully and had healthy offspring upon re-mating with another male of the same dose group (except one female at 100 mg/kg bw/day, which was not pregnant). No morphological abnormalities were seen in the reproductive organs which could account for the unsuccessful mating and in the absence of a dose-related trend, these cases of unsuccessful mating were considered unrelated to the test item.

Precoital time was not affected by treatment. Most females showed evidence of mating within four days of the first or second cohabitation period.
Two control females and one female at 1000 mg/kg bw/day showed evidence of mating after 13 days or after 6 days (upon re-mating), respectively. Based on the occurrence in the control group and as no abnormalities were seen in the reproductive organs of these rats, these longer precoital times were considered unrelated to treatment with the test item.


Fertility index was considered not to be affected by treatment. One control female, two females at 100 mg/kg bw/day and one female at 300 mg/kg bw/day were not pregnant. In the absence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment with the test item.

Gestation index and duration of gestation were considered not to be affected by treatment with the test item. All pregnant females had living pups on Day 1 of lactation, resulting in a gestation index of 100% for all groups.

Details on results (P0)

The number of implantation sites was considered not to be affected by treatment with the test item.
A slightly lower mean number of implantation sites was observed for females at 1000 mg/kg bw/day when compared with the concurrent control mean (i.e. 11.5 vs. 12.7). However, as individual values were within the normal range, this finding was considered unrelated to treatment with the test item.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of reproduction toxicity effects at dose levels up to and including the highest dose level tested

2

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of systemic toxicity at dose levels up to and including the highest dose level tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment with the test item.
Two pups of one litter at 100 mg/kg bw/day had breathing difficulties during the first litter check, one pup of another litter at 100 mg/kg bw/day had a partially paralyzed left hind leg from Day 7 of lactation onwards and two pups of a litter at 1000 mg/kg bw/day were cold and pale during the first litter check. These single occurrences were considered unrelated to treatment with the test item.
The nature and incidence of other observed clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Mortality / viability:

mortality observed, non-treatment-related

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 98, 98, 100 and 92% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Two pups of the control group (both from the same litter), 2 pups at 100 mg/kg bw/day (both from the same litter) and 9 pups at 1000 mg/kg bw/day (all from the same litter) were found dead at first litter
check. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability indices were 100% for the control and 100 mg/kg/day groups, and 99% for the 300 and 1000 mg/kg bw/day groups.
One pup at 300 mg/kg bw/day and one pup at 1000 mg/kg/day were missing on PND 3 and 2, respectively. These pups were most likely cannibalized. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was unaffected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.

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:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

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.

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 two male pups from the same litter at 100 mg/kg bw/day, one nipple was observed at PND 13. As for none of the other examined male pups at PND 13 nipples were observed, and this incidence in one litter of the low dose group did not show a dose-related trend, this finding was considered not to be related to treatment with the test item.

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 with the test item.
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 with the test item.

Details on results (F1)

Litter size was considered not affected by treatment with the test item.
Live litter sizes were 11.9, 11.5, 12.1 and 9.9 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
The lower mean live litter size at 1000 mg/kg/day was mostly attributed to the low number of living pups of a single female at first litter check (i.e. 2 alive and 9 dead pups). If this litter would not be taken into account, the mean live litter size of females at 1000 mg/kg bw/day would be 10.8, which is close to the historical control mean (historical control data of living pups at first litter check in Wistar Han rats (period 2016-2020): mean = 11; P5 – P95 = 6.0 – 15.0 (n=1445)).

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 96, 90, 94 and 94% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
A slightly lower survival index was noted for females at 100 mg/kg bw/day. This was mostly attributed to a single female which had 13 implantation sites but only 4 pups in total (2 alive
and 2 dead pups). Given the occurrence in a single low dose female only, this finding was
considered unrelated to treatment.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

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: Absence of developmental toxicity effects at dose levels up to and including the highest dose level tested.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Correlation of Histopathology Findings with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups

Group	Dose level mg/kg/day	Female/Male Nos.	In-Life Reason	Histopathology
1	0	- / 01	Not mated	-/-
		- / 03	Not mated	-/-
		49 / 09	Not pregnant	-/-
2	100	- / 14	Not mated	-/-
		- / 19	Not mated	-/-
		59 / 20	Not pregnant	-/-
		60 / 20	Not pregnant	-/-
3	300	62 / 22	Not pregnant	-/-
4	1000	-  / 39	Not mated	-/-
-        = not applicable.

Applicant's summary and conclusion

Conclusions:

In an oral screening study for reproductive and developmental effects, performed according to OECD guideline 421 and following GLP principles, both the parental and the reproduction NOAEL for Thickener (MDI-CHA-ODA) were derived to be ≥1000 mg/kg bw/day, based on the absence of adverse effects up to and including 1000 mg/kg bw/day.

Executive summary:

A 28-day screening study for reproductive and/or developmental effects was performed according to OECD/EC guidelines and GLP principles. Thickener (MDI-CHA-ODA) was administered by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The control group received the vehicle, corn oil, alone. Males were treated for 29-37 days, females that delivered offspring were treated for 50-68 days and females that failed to deliver pups were treated for 42-55 days.

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, male T4 thyroid hormone levels, macroscopic examination, organ weights, and microscopic examination).

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

In conclusion, the parental NOAEL as well as the NOAELs for reproduction and developmental effects were concluded to be at least 1000 mg/kg bw/day.