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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Remarks:
No deviations ocurred that negatively impacted the integrity of the study.
GLP compliance:
yes
Justification for study design:
SPECIFICATION OF STUDY DESIGN: Screening study conducted per OECD 422 (2016).

Test material

Constituent 1
Reference substance name:
Reaction products of fatty acids, C18-unstaturated, dimers and trimers with 3,3'-[oxybis(ethane-2,1-diyloxy)]dipropan-1-amine
EC Number:
701-270-9
Cas Number:
68911-25-1
Molecular formula:
N.A. UVCB substance
IUPAC Name:
Reaction products of fatty acids, C18-unstaturated, dimers and trimers with 3,3'-[oxybis(ethane-2,1-diyloxy)]dipropan-1-amine
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company
- Expiration date of the lot/batch: 31 January, 2021
- Purity test date: 19 December, 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: Stability is confirmed for at least 24 hours at room temperature under normal laboratory light conditions, at least 8 days in the refrigerator and at least 3 weeks in the freezer (≤ -15°C) over the concentration range at 0.965 mg/g (1 mg/mL) and 195 mg/g (200 mg/mL) (solutions)
- Solubility and stability of the test substance in the solvent/vehicle: Stability is confirmed for at least 24 hours at room temperature under normal laboratory light conditions, at least 8 days in the refrigerator and at least 3 weeks in the freezer (≤ -15°C) over the concentration range at 0.965 mg/g (1 mg/mL) and 195 mg/g (200 mg/mL) (solutions)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Stability is confirmed for at least 24 hours at room temperature under normal laboratory light conditions, at least 8 days in the refrigerator and at least 3 weeks in the freezer (≤ -15°C) over the concentration range at 0.965 mg/g (1 mg/mL) and 195 mg/g (200 mg/mL) (solutions)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item dosing formulations (w/w) were homogenized in propylene glycol to visually acceptable levels at appropriate concentrations to meet dose level requirements.

Test animals

Species:
rat
Strain:
Wistar
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 (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: 10-11 weeks, Females: 13-14 weeks
- Weight at study initiation: Males: 278-319 g, Females: 204-256 g
- Fasting period before study: None
- Housing: On arrival and following the pretest (females only) and pre-mating period, 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). Recovery males and females were
housed as such during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in
Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, Main females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 48-73
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 13 July, 2019 To: 14 November, 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized in propylene glycol to visually acceptable levels at appropriate concentrations to meet dose level requirements.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test article homogenization and test system compatibilit.
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Not specified
- Proof of pregnancy: Evidence of vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Duration of treatment / exposure:
Main males and Recovery males were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females were treated for 50-56 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 14 days after delivery, up to and including the day before scheduled necropsy. Main females which failed to deliver were treated for 42-53 days. Recovery females were treated for 51 Days.
Frequency of treatment:
Daily
Details on study schedule:
Main males and Recovery males were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females were treated for 50-56 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 14 days after delivery, up to and including the day before scheduled necropsy. Main females which failed to deliver were treated for 42-53 days. Recovery females were treated for 51 Days.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 - Control (Propylene glycol)
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
Groups 1 and 4: 15 (10 main study, 5 recovery)
Groups 2 and 3: 10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 15-day Dose Range Finder with oral administration of MTDID 18990 in rats
- Rationale for animal assignment (if not random): A total of 50 females was selected at randomization before initiation of the pretest phase. Out of 40 Main females, each female classified as not having regular estrous cycles during the pretest phase was replaced before initiation of dosing by one of the 8 additional females having regular estrous cycles, if feasible. A total of 40 females with regular estrous cycles and 10 Recovery females continued in the study. The supernumerary females were removed from the study, and their estrous cycle results were kept in the raw data but not reported. Animals were assigned to groups by a computer-generated random algorithm according to body weights, with all animals within ± 20% of the sex mean. Males and females were randomized separately. Animals in poor health or at extremes of body weight range were not assigned to groups.
Positive control:
NA

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main 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.
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 (Main and Recovery) during 14 days prior to treatment (pretest period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed. On the day of necropsy, a vaginal lavage was also taken from Main and Recovery females to determine the stage of estrous. This was done for all females, except for females that died spontaneously.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
Testes weight, seminal vesicle weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes: To reduce variability among the litters, on PND 4 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 AGD, 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
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, T4 thyroid hormone measurement

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Main group: Following completion of the mating period (a minimum of 28 days of administration). Recovery group: After the recovery period of at least 14 days, which is at least 14 days after the scheduled necropsy of Main males.
- Maternal animals: Main group females that delivered: PND 14-16. Main group females that failed to deliver: With evidence of mating: Post-coitum Days 27 (No. 68 (Group 2).
With evidence of mating: Approximately 26 days after the last day of the mating period (Nos. 56 (Group 1) and 86 (Group 4).
Without evidence of mating: Approximately 25 days after the last day of the mating period (No. 60 (Group 1)).
Recovery group females: After the recovery period of at least 14 days, which is at least 14 days after the first scheduled necropsy of Main females.

GROSS NECROPSY
-All animals (both Main and Recovery) were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were weighed at necropsy:
Brain
Cervix
Epididymis
Adrenal Gland
Coagulation Gland
Parathyroid Gland
Prostate Gland
Seminal vesicle
Thyroid
Heart
Kidney
Liver
Ovaries
Spleen
Testes
Thymus
Uterus

The following tissues were collected and examined microscopically:

Bone marrow
Bone, femur
Bone, sternum
Brain (eight levels)
Cervix
Epididymides
Eye
Gland, adrenal
Gland, coagulation
Gland, mammary
Gland, parathyroid
Gland, pituitary
Gland, prostate
Gland, seminal vesicle
Gland, thyroid
Gross lesions/masses
Gut-associated lymphoid tissue
Heart
Kidney
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Liver
Lung
Lymph node (mandibular and mesenteric site)
Muscle, skeletal
Nerve, sciatic
Ovaries
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord
Spleen
Stomach
Testes
Thymus
Trachea
Urinary bladder
Uterus
Vagina
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on PND 14-16.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy: 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. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (one male and one female pup).

HISTOPATHOLOGY / ORGAN WEIGTHS
None: Screening study
Reproductive indices:
The following indices were calculated:
Mating index
Percoital time
Fertility index
Gestation index
Duration of gestation
Offspring viability indices:
Post-implantation survival index
Live birth index
Percentage live males and live females at first litter check
Viability index
Lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No relevant clinical signs were noted up to 1000 mg/kg/day and no findings were noted during the weekly arena observations in this study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality was observed during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related changes in body weights and body weight gain were observed up to 1000 mg/kg/day.
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 up to 1000 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test article-related changes were identified in pupillary reflex up to 1000 mg/kg/day.
Haematological findings:
no effects observed
Description (incidence and severity):
Hematological parameters of treated rats were considered unaffected by treatment up to 1000 mg/kg/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant changes noted in clinical biochemistry parameters in males or females treated up to 1000 mg/kg/day at the end of the treatment or recovery periods.

The following statistically significant changes distinguished animals treated at 1000 mg/kg/day from control animals. Relative changes in mean values compared to the control group are indicated between parentheses.
• An increase in plasma alanine-aminotranferase (ALAT) concentrations (1.29x) in males at the end of the treatment period. Mean values remained within the historical control range1.
• An increase in plasma concentrations of inorganic phosphate (1.11x) in males at the end of the treatment period mean values remained within the historical control range1.
• An increase in total protein levels (1.06x) in non-lactating females at the end of treatment.
• A decrease in total bilirubin concentrations (0.88x) in non-lactating females at the end of treatment.
• An increase in glucose concentrations in non-lactating females at the end of treatment (1.30x) and recovery (1.17x).

As ALAT and inorganic phosphate values (and most individual values) in 1000 mg/kg/day males remained within the historical control ranges1 and at the magnitude of the changes for the non-lactating females, changes were considered not toxicologically relevant. Changes in inorganic phosphate values (and most individual values) were considered unrelated to treatment in absence of a dose related response.

Thyroid hormone analyses:
Serum levels of total Thyroxine (T4) in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No adverse functional findings were observed during the study.
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):
No adverse microscopic findings were noted during the study.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No adverse microscopic findings were noted during the study.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
Description (incidence and severity):
No histopathological findings were noted in the epididymides.
Reproductive performance:
no effects observed
Description (incidence and severity):
No reproductive parameters were impacted by treatment with the test article.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (sperm measures)
reproductive performance

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.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality 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 indices were 96, 100, 99 and 99% for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
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.
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:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female 14-16 pups were considered not to be affected by treatment with the test item.
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 macroscopic findings were noted among pups that were considered to be related to the test item.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
No reproductive or developmental parameters were adversely impacted by treatment up to 1,000 mg/kg/day. No parental toxicity was observed in any of the parameters examined up to the highest dose level tested 1000 mg/kg/day. Based on the results of the study, the repeated dose, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of MTDID 18990 was 1,000 mg/kg/day.
Executive summary:

The repeated dose and reproductive/developmental toxicity potential of MTDID 18990 was evaluated in Wistar rats. The study was conducted according to OECD 422 in compliance with OECD GLP regulations. Male and female Wistar Han rats were treated with MTDID 18990 suspended in propylene glycol by daily oral gavage at dose levels of 0 (vehicle control) 100, 300 and 1000 mg/kg/day, followed by a 14-day treatment-free recovery period. 15 animals per sex per dose were utilized in the control and 1000 mg/kg/day groups with 5 included in the recovery period. 10 animals per sex per dose were utilized in the 100 and 300 mg/kg/day groups. The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproductive or developmental toxicity. Main group males and recovery males were treated for 29 days, including a minimum of 14 days prior to mating and during the mating period for Main males. For both Main and Recovery male’s treatment ended one day before scheduled necropsy of Main males. Main females were treated for 50-56 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 14 days after delivery, up to and including the day before scheduled necropsy. Main females which failed to deliver were treated for 42-53 days. Recovery females were treated for 51 Days. Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy. Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated Main 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. Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pretest period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed. On the day of necropsy, a vaginal lavage was also taken from Main and Recovery females to determine the stage of estrous. thyroid hormone t4 (F0 males), gross necropsy findings, organ weights and histopathological examinations were performed. The following reproductive/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). At microscopic examination an increase in trabecular bone was observed in males at 1000 mg/kg/day. This is an unusual finding and was observed at an increased incidence and severity in the bone-femur of males at 1000 mg/kg/day. This finding was also present at minimal degree in a single male of the recovery control group at Day 32 of treatment. This alteration was not seen in the sternum or tibia of the affected males and was not present in females. Based on the low severity and absence of any accompanying alterations, the increase in trabecular bone was considered non-adverse. No reproductive or developmental parameters were adversely impacted by treatment up to 1,000 mg/kg/day. No parental toxicity was observed in any of the parameters examined up to the highest dose level tested 1000 mg/kg/day. Based on the results of the study, the repeated dose, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of MTDID 18990 was 1,000 mg/kg/day.