Reactionmass of dodecan-2-yl prop-2-enoate and dodecan-3-yl prop-2-enoate and dodecan-4-yl prop-2-enoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July, 2016

Deviations:

no

Remarks:

No deviations occurred that negatively impacted the results of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reactionmass of dodecan-2-yl prop-2-enoate and dodecan-3-yl prop-2-enoate and dodecan-4-yl prop-2-enoate

EC Number:

947-818-2

Cas Number:

1612783-92-2

Molecular formula:

C15H28O2

IUPAC Name:

Reactionmass of dodecan-2-yl prop-2-enoate and dodecan-3-yl prop-2-enoate and dodecan-4-yl prop-2-enoate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot 6
- Expiration date of the lot/batch: 04 October, 2019
- Purity test date: 04 October, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area set to maintain 18°C to 24°C
- Stability under test conditions: Stable per analytical verification of test article formulations in corn oil
- Solubility and stability of the test substance in the solvent/vehicle: Soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Non-reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was prepared in corn oil

FORM AS APPLIED IN THE TEST: Prepared in corn oil.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control 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 Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 203-395 g
- Fasting period before study: None
- Housing: On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order.
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 12 June, 2018 To: 10 August, 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test article was dissolved in corn oil.

VEHICLE
- Justification for use and choice of vehicle: Test article solubility and test system tolerance.
- Concentration in vehicle: 8, 30, or 140 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): 2GK0223
- Purity: No data

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of mating was confirmed via copulatory plug or sperm in a vaginal lavage.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No, only one attempt at mating was conducted.
- 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 (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Duplicate sets of samples (1.0 mL) from the first preparation were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤ 10% and if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup
samples were discarded.

Duration of treatment / exposure:

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating and continuing through mating, for a minimum of 28 days, until 1 day prior to euthanasia. Females were dosed for 14 days prior to mating and continuing through Lactation Day 12. All animals were dosed at approximately the same time each day

Frequency of treatment:

Daily

Details on study schedule:

Males were dosed for 14 days prior to mating and continuing through mating, for a minimum of 28 days, until 1 day prior to euthanasia. Females were dosed for 14 days prior to mating and continuing through lactations Day 12. All animals were dosed at approximately the same time each day. The following parameters and endpoints were evaluated in this study: clinical signs, body weights, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, thyroid hormones, gross necropsy findings, organ weights, and histopathology. At necropsy, the adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries (with oviducts), pituitary gland, prostate gland, seminal vesicle (with coagulating gland and fluid), spleen, testes, thymus gland, and thyroid were weighed. Tissue samples of the brain, coagulating gland, kidneys, liver, mammary glands, ovaries with oviducts, pituitary gland, prostate gland, seminal vesicles (2), testes with epididymides (2), vas deferens, thyroid with parathydroids (2), uterus with cervix and vagina, and gross lesions were collected at necropsy from all animals. Histopathological examination was performed on the tissue samples collected (except the liver, kidney, and thyroid) from animals in the control and high-dose groups and gross lesions were examined from all groups. 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control (Corn Oil)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

700 mg/kg bw/day

No. of animals per sex per dose:

10

Details on study design:

- Dose selection rationale: The dose levels were selected based on a 28 day study conducted on a structural analog.
- Rationale for animal assignment (if not random): Random

Positive control:

None

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, once in the morning and once in the afternoon. Animals were not removed from cage
during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were removed from the cage, and a detailed clinical observation was performed once daily throughout the study. During the dosing period, these observations were performed
prior to dosing. On dosing days, clinical observations were also recorded approximately 4 hours postdose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually bi-weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (if possible), 1, 4, 7, 10, and 13.

Oestrous cyclicity (parental animals):

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of
mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations:
testis weight, epididymis weight, seminal vesicles

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No - screening study

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, throid hormone evaluation.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed on Study Day 30.
- Maternal animals: All surviving animals were sacrificed on Lacatation Day 13.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were weighed at necropsy:

Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries (with oviducts)
Pituitary gland
Prostate gland
Seminal vesicle (with coagulating gland and fluid)
Spleen
Testes
Thymus gland
Thyroid (with parathyroids)

The following tissues were collected and examined microscopically:

Brain
Coagulating gland
Kidneys
Liver
Mammary glands
Ovaries with oviducts (2)
Pituitary gland
Prostate gland
Seminal vesicles (2)
Testes with epididymides (2) and vas deferens
Thyroid with parathyroids (2)
Uterus with cervix and vagina
Gross lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at Postnatal Day (PND) 13
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. All other animals were discarded without examination.

GROSS NECROPSY
On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. All other animals were discarded without examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
Representative specimens with malformations from offspring found dead or euthanized for humane reasons were preserved in 10% neutral buffered formalin. Thyroid were weighed at necropsy.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals or cages (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit. Statistical analyses were not conducted on F0 bi-weekly female body weight data after 1 or more animals had entered the gestation phase. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Parental and offspring body weights and body weight changes, parental food consumption, estrous cycle lengths, precoital intervals, gestation lengths, former implantation sites, unaccounted-for sites, live litter size on PND 0, numbers of pups born, absolute and relative organ weights, thyroid hormone values, anogential distance (absolute and relative to the cube root of body weight), and number of nipples/areolae values were subjected to a parametric one-way ANOVA10 to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Histopathological findings of each treated group were analyzed using Fisher’s Exact Test.

Reproductive indices:

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of
mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Parental mating, fertility, copulation, and conception indices, precoital intervals, gestation lengths, former implantation sites and unaccounted-for sites were all calculated or recorded.

Offspring viability indices:

Live litter size, numbers of pups born, sex percentages at birth, postnatal survival.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical findings were noted.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All F0 animals survived to the scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight gains in the 40, 150, and 700 mg/kg/day group animals were unaffected by test substance administration throughout the study. None of the differences from the control group were statistically significant.

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day, in the 40, 150, and 700 mg/kg/day group males and females was similar to that in the control group throughout the study. No statistically significant differences were observed.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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):

There were no test substance-related histologic changes.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no test substance-related histologic changes.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean lengths of estrous cycles in these groups were similar to the control group value.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test article-related changes were noted in testis weight, epididymis weight, seminal vesicles.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. All males sired a litter and all females were gravid.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

700 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

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

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:

effects observed, non-treatment-related

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance administration.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Three (2), 2(2), 10(4), and 5(5) pups (litters) in the control, 40, 150, and 700 mg/kg/day groups, respectively, were found dead or euthanized in extremis. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead or euthanized in extremis. Aside from the presence or absence of milk in the stomach, the only internal findings were noted for a single pup (No. 5707-01) in the 40 mg/kg/day group. This pup had malformations of cyclopia, vertebral anomaly without associated rib anomaly, and costal cartilage anomaly. In addition, variations of vertebral centra not fully ossified and distended ureters were noted for this pup. However, these findings did not occur in a dose-related manner, and therefore were not considered test substance-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes during PND 1–13 in the 40, 150, and 700 mg/kg/day groups were unaffected by parental administration of the test substance.
No statistically significant differences from the control group were noted.

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):

There were no test substance-related effects on serum levels of T4 (thyroxine), or TSH (thyroid stimulating hormone) in F1 males and females at any dosage level.

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 internal findings were noted at the necropsy of pups euthanized on PND 13.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. The test substance-treated group values were not statistically different from the control group values.

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 40, 150, and 700 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

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:

700 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:

Under the conditions of this study, due to the absence of adverse effects at any dosage level, a dosage level of 700 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity of MTDID 44430 when administered orally by oral gavage to rats.

Executive summary:

The objective of this study was to provide data on the potential effects of MTDID 44430 on reproductive performance, pup development, and general toxicity of rats by oral exposure. The study was conducted according to OECD 421 under OECD GLP conditions.  Four groups of rats (n= 10/sex) received 0 (control), 40, 150, and 700 mg/kg MTDID 44430 in corn oil as a single daily oral gavage dose. Males were dosed for 14 days prior to mating and continuing through mating, for a minimum of 28 days, until 1 day prior to euthanasia. Females were dosed for 14 days prior to mating and continuing through lactations Day 12. All animals were dosed at approximately the same time each day. The following parameters and endpoints were evaluated in this study: clinical signs, body weights, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, thyroid hormones, gross necropsy findings, organ weights, and histopathology. At necropsy, the adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries (with oviducts), pituitary gland, prostate gland, seminal vesicle (with coagulating gland and fluid), spleen, testes, thymus gland, and thyroid were weighed. Tissue samples of the brain, coagulating gland, kidneys, liver, mammary glands, ovaries with oviducts, pituitary gland, prostate gland, seminal vesicles (2), testes with epididymides (2), vas deferens, thyroid with parathydroids (2), uterus with cervix and vagina, and gross lesions were collected at necropsy from all animals. Histopathological examination was performed on the tissue samples collected (except the liver, kidney, and thyroid) from animals in the control and high-dose groups and gross lesions were examined from all groups. 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. RESULTS: All F0 animals survived to the scheduled necropsy. No test substance-related clinical findings were noted. There were no statistically significant changes in mean body weight, mean food consumption, reproductive performance, parturition, thyroid hormones, anogenital distance, areolate/nipple anlagen, litter viability and survival. There were no test substance-related changes in organ weight, gross pathology, and histopathology. No internal findings were noted at the necropsy of euthanized pups. Under the conditions of this study, due to the absence of adverse effects at any dosage level, a dosage level of 700 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity of MTDID 44430 when administered orally by oral gavage to rats.