Registration Dossier
Registration Dossier
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EC number: 205-528-7 | CAS number: 142-22-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Effects on fertility
Description of key information
- Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422, GLP), dermal, 0, 150, 454, and 1030 mg/kg/day, rat: NOAEL parental toxicity 1030 mg/kg/day, NOEL reproductive performance was 1030 mg/kg/day
- Extended One-Generation Reproductive Toxicity Study (OECD 443, GLP), oral, 0, 25, 60 and 115 mg/kg/day, rat, NOAEL (general toxicity, F0) 25 mg/kg/day, NOAEL (general toxicity, F1) could not be established as adverse findings were noted in the liver starting at 25 mg/kg/day, NOAEL (reproduction, F0) >=115 mg/kg/day, NOAEL (reproduction, F1) 60 mg/kg/day (based on irregular estrous cycles and lower sperm counts; findings were noted at the high dose in which also systemic toxicity was noted including three F0-mortalities), NOAEL (development) >= 115 mg/kg/day
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Remarks:
- Combined Reproduction/Developmental Screening study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- August 12, 2003-October 20, 2003
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan
- Age at study initiation: eight weeks (six weeks at arrival plus two-week acclimation period)
- Weight at study initiation: All animals placed on study had body weights within ±20% of the mean body weight for each sex. Forty male rats (weighing 223 to 292 g at randomization) and 80 female rats (weighing 163 to 205 g at randomization)
- Fasting period before study: No
- Housing: Animals were individually housed in stainless steel cages with wire mesh fronts and bottoms, suspended over pans containing absorbent liners, except during pairing, near parturition, and during lactation (see details on mating procedure).
- Diet (e.g. ad libitum): yes, except during designated fasting periods
- Water (e.g. ad libitum): yes. Tap water was available ad libitum to all animals and supplied via an automatic watering system, except while females were housed in plastic cages for parturition and lactation. During this time, water was supplied using glass bottles attached to each cage.
- Acclimation period: two weeks
ENVIRONMENTAL CONDITIONS
Temperature and relative humidity in the animal room were monitored and recorded daily
- Temperature (°C): 67 to 74°F
- Humidity (%): 21 to 69%
- Air changes (per hr): data not available
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided for approximately 12 hours per day via an automatic timer. - Route of administration:
- dermal
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- TEST SITE
- Area of exposure: Test areas: an anterior and posterior site on the back. The dose sites were alternated daily between the anterior and posterior areas.
- % coverage: no less than 10% of the total body surface.
- Type of wrap if used: The control and test article were drawn into a plastic 1 mL syringe (except for the 150 mg/kg/day groups, which required a 100 µL glass syringe), administered from the hub of the syringe, and distributed evenly over the appropriate site.
- Time intervals for shavings or clipplings: at weekly intervals throughout the study, all animals had hair clipped from the test areas
REMOVAL OF TEST SUBSTANCE
- Washing (if done): the sites were washed with warm soapy water, and rinsed with tepid tap water.
- Time after start of exposure: six hours after test article administration
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at dose levels of 150, 454, and 1030 mg/kg/day at respective dose volumes of 0.13, 0.4, and 0.9 mL/kg.
- Concentration (if solution): not applicable
- Constant volume or concentration used: yes. The dose volumes were derived on the basis of the density of the test article, 1.143 grams/mL.
CONTROL MATERIAL
- Amount(s) applied (volume or weight with unit): based on the most recent body weightt at dose volume of 0.9 mg/mL
- Concentration (if solution): not applicable
- Lot/batch no. (if required): 03-144-JT-03, 04-146-JT, 04-100-JT
- Purity: Documentation on the strength, purity, composition, stability, physical properties, and other pertinent information on each batch of control article (0.9% Sodium Chloride for Injection, USP) was limited to that information listed on the label of this commercially available control article.
USE OF RESTRAINERS FOR PREVENTING INGESTION: yes. Immediately following dosing, the application site was covered with gauze dressing and secured with non-irritating tape. Six hours after test article administration, the gauze and tape were removed, the sites washed and rinsed. The dose site of each animal was blotted dry prior to returning to the cage. - Details on mating procedure:
- - M/F ratio per cage: 1
- Length of cohabitation: After two weeks of treatment, the males were randomly cohabited nightly, one male with one female, with corresponding females in the same treatment or control group. Animals were paired for mating in the cage of the male. The same males and females were cohoused nightly until mating occurred or for a maximum of 14 days.
- Proof of pregnancy: [vaginal plug ] referred to as [day 0 ] of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no
- Further matings after two unsuccessful attempts: no
Each day during the mating period, the female was removed from the cage of the male until after the six-hour exposure period, at which time the female was placed back into the cage of the male.
- After successful mating each pregnant female was caged (how): When evidence of mating was noted, the female was removed from the cage of the male and individually housed for the remainder of gestation. After the mating period, any unmated females were individually housed in wire mesh cages. Any of these females that appeared to be pregnant were continued on treatment until it was determined that treatment should stop, at which time, the females were transferred to a solid plastic cage with wood chip bedding in anticipation of parturition.
- Any other deviations from standard protocol: no - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- A compositional analysis was conducted on test article samples (2 g/sample) collected prior to initiation of dosing (start of study) and after completion of test article administration (end of study). The analysis was performed to confirm the identity of the test article by HPLC TAN and NMR procedures.All compositional analytical work was conducted by Ricerca, LLC, Concord, Ohio. The work performed by Ricerca in conjunction with this study was conducted in compliance with GLP regulations and subjected to review by the Quality Assurance Unit (QAU) of that laboratory.
- Duration of treatment / exposure:
- up to 48 days in females in the reproductive component, depending on reproductive performance (14 days premating, up to 14 days of mating, and through Day 20 of gestation)
- Frequency of treatment:
- once per day at approximately the same time each day
- Details on study schedule:
- - Age at mating of the mated animals in the study: 10 weeks: (six week at arrival plus two-week acclimation period plus two weeeks premating period)
- Dose / conc.:
- 150 mg/kg bw/day
- Dose / conc.:
- 454 mg/kg bw/day
- Dose / conc.:
- 1 030 mg/kg bw/day
- No. of animals per sex per dose:
- - ten male and ten female at dose levels of 150, 454 and 1030 mg/kg bw/day in repeat dose component
- ten female at dose levels of 150, 454 and 1030 mg/kg bw/day in reproductive component. The ten male animals of each repeat dose group were also utilized for the reproductive/development component of the study. - Control animals:
- other: (0.9% sodium chloride for injection U.S.P.)
- Details on study design:
- - Dose selection rationale: The dose levels were selected in consultation with the Sponsor, on the basis of available data from previous studies.
- Rationale for animal assignment (if not random): randomized
- Other: Control group, treated only with vehicle alone, was needed to exclude any effect of vehicle and make possible to interpret the effects of test material - Positive control:
- No
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once during test or control article administration, and once following completion of the six-hour exposure and removal of the wrap) for morbidity, mortality, signs of injury, and the availability of food and water.
- Cage side observations checked in table [No.1] were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of treatment and weekly during the study.
Deatiled clinical observations checked in table [No.1] were included
BODY WEIGHT AND BODY WEIGHT GAIN: Yes
- Time schedule for examinations: the day after arrival, at randomization, at initiation of test or control article administration, weekly during the study, and at termination. Females in the reproductive component were weighed on Days 0, 7, 14, and 20 of gestation and females that delivered were weighed on Days 0 and 4 of lactation. Females in the reproductive component that did not mate continued to be weighed weekly until delivery (undetermined pregnancies) or scheduled euthanasia.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No, but in g food/animal/day
Individual food consumption for males was measured and recorded weekly during the study, except during the two-week mating period. Food consumption was also measured weekly during the premating period for reproductive females. During the mating period, food consumption was not recorded because the animals were cohabited nightly. Food consumption was measured and recorded for females in the reproductive component on Days 0, 7, 14, and 20 of gestation. Food consumption for unmated females was generally recorded for one week following completion of the mating period. For females with litters, food consumption was measured and recorded for Days 0 and 4 of lactation. Food consumption was calculated for Days 0-7, 7-14, and 14-20 of gestation, and Days 0-4 of lactation.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No - Oestrous cyclicity (parental animals):
- not examined
- Sperm parameters (parental animals):
- not examined
- Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [no] On Day 0. The litters were examined as soon as possible after delivery
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded. No . All pups of the litters were examined.
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups (litter size), sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight, physical or behavioural abnormalities, other: clinical observations (pelage/skin-abrasions, hind limb, skin colour ] on day 0 and 4 of lactation
GROSS EXAMINATION OF DEAD PUPS:
[ yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.] - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.] After seven weeks of treatment)
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.] On postnatal day 4
GROSS NECROPSY were performed for all males and females.
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] All tissues (Table 2)
HISTOPATHOLOGY were performed only for males / ORGAN WEIGHTS were performed for all males and all females
The tissues indicated in Table [2] were prepared for microscopic examination and weighed, respectively. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring animals were sacrificed at [4] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: only macroscopic (all external tissues)
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes, only external examined
HISTOPATHOLOGY / ORGAN WEIGTHS
not examined - Statistics:
- Group Pair-wise Comparisons
For each specified endpoint and for all collection intervals, Levene's test 5 was used to assess homogeneity of group variances. If Levene's test was not significant (p>0.01), Dunnett's test6 was used to compare each treatment group with the control group. If Levene's test was significant (p<0.01), comparisons with the control group were made using Welch's t-test7 with a Bonferroni correction. Results of all pair-wise comparisons were reported at the 0.05 and 0.01 significance levels. All endpoints were analyzed using two-tailed tests. Besides these tests, Log Transformation, Fisher's Exact Test, Covariate Analysis and Arcsin-Square-Root Transformation were used. - Reproductive indices:
- Male and female fertility and mating indices were calculated as percentages of pregnant or delivered animals
- Offspring viability indices:
- Pup survival indices during lactation were calculated as percentages of liveborn pups per litter for every dose group
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- not examined
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 1 030 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- 1 030 mg/kg bw/day (nominal)
- Sex:
- male/female
- Basis for effect level:
- other: No treatment related effects were found.
- Reproductive effects observed:
- not specified
- Conclusions:
- In this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated. The No-Observable-Effect-Level (NOEL) of reproductive performance was 1030 mg/kg/day, the highest dose level evaluated.
- Executive summary:
This study was conducted in accordance with OECD Guideline 422, which is comprised of two components, a repeat dose toxicity study with neurobehavioral evaluations and a reproduction/developmental toxicity screening (see also section Repeated dose toxicity: dermal).
The purpose of the reproduction/developmental toxicity screening component was to provide information on possible effects on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. Both the repeat dose and the reproductive/developmental component were comprised of three treatment groups and a saline-treated control group. Each reproductive group contained ten female Sprague-Dawley rats) [Crl: CD" (SD)IGS BR]. The ten male animals of each repeat dose group were also utilized for the reproductive/developmental component of the study. Animals were administered dermally once daily via occlusion for 6 hours at dose levels 150, 454, and 1030 mg/kg/day for at least 42 consecutive days, while females in the reproductive component were treated for two weeks before pairing, during pairing, and from Gestation Days (GD) 0 to 20. The control animals received 0.9% Sodium Chloride, USP, at a volume of 0.9 mL/kg for the same duration as the treated animals. After two weeks of treatment, the animals were cohabited nightly with males from the repeat dose component, one male to one female, from the same treatment group, for up to 14 days. Females were evaluated daily for evidence of mating (sperm in the vaginal rinse or vaginal plug). Once mating was confirmed (GD 0), females were separated from the male for the remainder of gestation, and allowed to deliver and nurse litters until Postnatal Day (PND) 4. Litter size and pup evaluations (body weight, sexing, and external examination) were recorded at birth and PND 4. Pups were euthanized and externally examined on PND 4 and the carcasses were discarded without further examination. Complete necropsies were performed on all parent animals (repeat dose and reproductive components) and organs and tissues were collected, weighed, and preserved.
No effect of treatment was evident from mortality, clinical evaluations, dermal evaluations, body weights, food consumption, organ weights, macroscopic or microscopic evaluations, reproductive performance, gestation and lactation body weights or food consumption, gestation length, litter size, pup body weight, pup sex ratios, or pup external examinations to PND 4. Thus, in this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated. The No-Observable-Effect Level (NOEL) of reproductive performance was 1030 mg/kg/day, the highest dose level evaluated.
- Endpoint:
- extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19 FEB 2020 to 25 FEB 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
- Version / remarks:
- June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Justification for study design:
- SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 10 weeks
- Basis for dose level selection: An oral 90-day repeated dose toxicity study and a Reproduction/Developmental Toxicity Screening Test, both in rats (please refer to cross references)
- Inclusion of extension of Cohort 1B: No
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B: No
- Inclusion of developmental immunotoxicity Cohort 3: No
- Route of administration: oral gavage
- Other considerations:
Justification for Test System and Number of Animals:
- 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. The Laboratory 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.
- The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. - 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.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks old
- Weight at study initiation: (P) Males: 158 - 211 g; Females: 121 - 168 g; (F1) Males: 41 - 66 g; Females: 39 - 61 g
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, type IV; height 18 cm or type 2000P; 61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm). During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21).
The cages contained appropriate bedding and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records. Animals were separated during designated procedures/activities.
- Diet (e.g. ad libitum): Pelleted rodent diet was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 14 day
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24°C
- Humidity (%): 46 to 68 %
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
IN-LIFE DATES: From: 03 Mar 2020 (initiating of dosing) To: 15 Sep 2020 (date of last necropsy) - Route of administration:
- oral: gavage
- Vehicle:
- polyethylene glycol
- Remarks:
- Polyethylene glycol 400
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
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 weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial preparations (not part of this study)
- Concentration in vehicle: 0, 5, 12, 23 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days was allowed for mating
- Proof of pregnancy: by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as Day 0 post-coitum.
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages
- Any other deviations from standard protocol:
Female No. 183 was initially paired with Male No. 83. This male was euthanized for humane reasons on Day 6 of the mating period. As the female had not shown evidence of mating, she was cohabitated with Male No. 81 of the same dose group.
For two couples (Male No. 26, Female No. 126 and Male No. 56, Female No. 156), detection of mating was not confirmed in first instance. The actual mating date was determined based on a re-evaluation of the vaginal lavage for presence of sperm cells. Consequently, these couples were separated 1 day after the actual mating date. The actual mating date was designated Day 0 post-coitum.
Detection of mating was not confirmed in first instance for Female Nos. 108 and 110. 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 continuation of di-estrus during the mating in this female. The mating date of these animals 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:
- Chemical analyses of formulations were conducted at three occasions during the study to assess accuracy and homogeneity. The Analyses were performed using a validated analytical procedure.
- Concentration Analysis: 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.
- Homogeneity Analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <= 10%.
- Stability Analysis: Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. - Duration of treatment / exposure:
- F0 males: 11-12 weeks (including 10 weeks pre-mating); females: 15-18 weeks (including 10 weeks pre-mating); females which failed to deliver or had a total litter loss: 13-14 weeks
F1: Cohort 1A: 10-11 weeks; Cohort 1B: 11-12 weeks; Cohort Surplus: Not applicable - Frequency of treatment:
- Once daily, 7 days a week
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Control
- Dose / conc.:
- 25 mg/kg bw/day
- Dose / conc.:
- 60 mg/kg bw/day
- Dose / conc.:
- 115 mg/kg bw/day
- No. of animals per sex per dose:
- F0: 25 / sex / dose group
F1: Cohort 1A: 20 / sex / dose group; Cohort 1B: 20 / sex / dose group; Surplus: 40 / sex / dose group (F1-animals of Cohort Surplus were not dosed) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The dose selection is based on the results of an oral 90-day repeated dose toxicity study and a Reproduction/Developmental Toxicity Screening Test, both in rats (please refer to cross references). Based on the findings noted in the above mentioned studies 25, 60 and 115 mg/kg/day were chosen as dose levels for the EOGRTS study.
- Rationale for animal assignment (if not random):
F0-animals were randomly assigned to groups at arrival. Males and females were randomized separately.
F1-animals: On PND 21, pups from available litters per group were selected and assigned according to the following schedule. 20 females with 8 live pups/litter were selected (if possible).
- Fasting period before blood sampling for clinical biochemistry: The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. - Positive control:
- no positve control
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: Animals were observed for general health/mortality and moribundity
CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily up to the day prior to necropsy.
These observations were at least conducted prior to dosing and 0-30 minutes after dosing.
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, 14 and 21. In order to monitor the health status, Male No. 83 (115 mg/kg/day) and Female No. 199 (115 mg/kg/day) were also weighed on Days 77 and 93 of treatment, respectively. 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, 14 and 21.
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.
OTHER:
- Arena Observations: Clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period.
- General Reproduction Data: From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
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. - Oestrous cyclicity (parental animals):
- Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating 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 scheduled necropsy, a vaginal lavage was also taken. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously. - Sperm parameters (parental animals):
- Parameters examined in all surviving F0 parental generations:
epididymis weight, sperm count in epididymides, sperm motility, sperm morphology - 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. 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:
- In-life Procedures, Observations, and Measurements – until weaning (PND 21): Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, clinical observations at least once daily for all pups, checked twice daily for general health/mortality.
- In-life Procedures, Observations, and Measurements – from Weaning (PND 21) onwards: Cohorts 1A and 1B: mortality/moribundity checks, clinical observations, arena observations, body weights, food and water consumption, vaginal patency, balanopreputial separation, stage of estrus determination; Cohort 1A: estrous cycle determination
GROSS EXAMINATION OF DEAD FETUSES and PUPS:
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally, if possible) and externally examined with emphasis on developmental morphology. For pups found dead from PND 14 onwards a limited necropsy was performed including sex determination (both externally and internally, if possible). Descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals, after successful mating and a minimum of 10 weeks of treatment.
- Maternal animals: All surviving animals, on LD 23-25, or after total litter loss, or failure to deliver, or within 24 hours after the last pup was found dead or missing. Except for females with total litter loss, all animals surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available.
GROSS NECROPSY
- 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 was recorded in addition.
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the corresponding table under 'Any other information' were prepared for microscopic examination and weighed, respectively.
OTHER:
Clinical Pathology:
Blood of 10 selected animals/sex/group of F0-animals was collected on the day of scheduled necropsy. The selected F0-animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Hematology, coagulation, clinical chemistry, thyroid hormone was examined. For analysed parameters, please refer to the corresponding table under 'Any other information'. - Postmortem examinations (offspring):
- SACRIFICE
- Age at Necropsy of F1 offsprings: Cohort 1A: PND 89-95; Cohort 1B: ≥ PND 97; Cohort Surplus: PND 22-24
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY
- Necropsy was done for Cohort 1A, 1B, Surplus and spare F1-animals. Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally, \if possible). Descriptions of all external abnormalities were recorded.
1. Cohort 1A animals were deprived of food overnight (with a maximum of 24 hours) before necropsy, but water was available. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
- For all males of Cohort 1A, the following assessments were performed: Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy.
Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (right) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing, the right epididymis was weighed, homogenized and evaluated for
sperm numbers. Evaluation was performed for all samples.
- From 10 selected animals/sex/group of Cohort 1A, splenic lymphocyte subpopulation analysis was performed at termination. If possible, one pup (male or female) was selected per litter (20 litters in total). The following subpopulations were determined in isolated splenic lymphocytes: T-cells, T-helper cells, T-cytotoxic cells, B-cells, NK-cells, Ratio T-helper cells/ T-cytotoxic cells (Th/Tc).
2. Cohort 1B animals and Cohort Surplus animals were not deprived of food overnight before necropsy. All animals were subjected to a limited examination, with special attention being paid to the reproductive organs.
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in corresponding table under 'Any other information' were prepared for microscopic examination and weighed, respectively.
OTHER:
Clinical Pathology: Hematology, coagulation, clinical chemistry, thyroid hormone was examined. For details on analysed parameters, please refer to the corresponding tables under 'Any other information'.
Blood Sampling:
- Cohort 1A : Blood of 10 selected animals/sex/group1 of Cohort 1A animals was collected on the day of scheduled necropsy. The selected Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Evaluation: hematology, coagulation, clinical chemistry, thyroid hormone examinations.
- Culled pups: On PND 4 at culling, blood was collected from two surplus pups per litter (≤ 25 litters/group). Evaluation: thyroid hormone analysis
- Cohort Surplus on PND 22: On PND 22-24, blood was collected from all Cohort Surplus animals (10/sex/group). Blood samples were collected into serum tubes for measurement of thyroid-stimulating hormone (TSH) and thyroxine (T4). - 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 according to sex and occasion. Descriptive statistics number, mean and standard deviation 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. Pairwise comparisons were made between the groups.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test). The follicle count data set (at least 3 groups) was compared using an overall Kruskal-Wallis. Whenever, the overall test is significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant. - Reproductive indices:
- Mating Index, Fertility Index, Gestation Index, Post-Implantation Survival Index, Weaning Index
- Offspring viability indices:
- Live Birth Index, Viability Index
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No toxicologically relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during weekly arena observations.
Salivation seen after dosing among animals of all dose groups during the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item and vehicle rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period 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. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- A total of four animals were sacrificed prematurely. Details on clinical signs, macroscopic and microscopic observations noted for animals sacrificed in extremis are described in the following:
Male No. 62 (treated at 60 mg/kg/day) was sacrificed in extremis on Day 66 as quick breathing, hunched posture, piloerection and partly closed eyes were noted directly after dosing. Based on these clinical signs in combination with the time point of occurrence (directly after dosing) a misdosing was suspected. At necropsy, many grey-white foci were noted on the lungs. No microscopic findings were noted that could explain the moribund condition of this animal and therefore a relationship with treatment with the test item could not be excluded.
Male No. 83 (treated at 115 mg/kg/day) presented with piloerection, diarrhoea, red excretion from the anus on Day 77, and a body weight loss of 15% compared to a week before and was therefore sacrificed for animal welfare reasons. At necropsy, an irregular surface of the liver was noted and the prostate, seminal vesicles and thymus were reduced in size. Microscopically, moderate necrosis together with basophilia/karyocytomegaly, peribiliary infiltration and bile duct hyperplasia in the periportal areas of the liver (with corresponding irregular surface of the liver) were observed. The lymphoid depletion and reduced size of the thymus and the decreased content/atrophy with corresponding reduced size of the seminal vesicles and prostate were considered secondary to the poor condition of the animal.
Female No. 199 (treated at 115 mg/kg/day) was pale, lethargic, had a decreased locomotor activity, a flat posture and piloerection on Day 93 (post-coitum Day 21). Based on its condition it was expected that she would not manage to deliver a healthy litter and was therefore sacrificed in extremis. Microscopically, massive necrosis in the liver (with corresponding of enlargement of the liver and yellow-reddish-black-brown foci on the liver), marked glomerulopathy with massive tubular necrosis in the kidneys (with corresponding enlargement of the kidney) and marked extra-medullary hematopoiesis were observed. In addition, in the uterus of this female dead pups were found which could explain all the findings mentioned but it is unclear whether the liver lesions were the cause or the result of the dead pups.
Female No. 182 (treated at 115 mg/kg/day) was sacrificed in extremis on Day 95 (lactation Day 1). After delivering the litter, this animal was pale, lethargic and piloerection and hunched posture were noted. Furthermore, she did not clear herself nor the pups after giving birth, and no/only a little milk was seen in the stomach of the pups. Microscopically, massive necrosis together with karyocytomegaly in the periportal areas of the liver (with corresponding irregular surface and yellow-reddish-black-brown foci on the liver) and marked glomerulopathy with marked tubular necrosis in the kidneys (with corresponding irregular surface) were noted.
Female No. 156 (treated at 60 mg/kg/day) was sacrificed on Lactation Day 1 as she had a total litter loss. - Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A reduced body weight gain (up to 0.86x of control) and lower body weights (up to 0.90x of control) were noted for males treated at 115 mg/kg/day from Week 5 onwards (not always statistically significant).
During the pre-mating, mating, post-coitum and lactation periods higher body weights (up to 1.06x of control) were noted for females treated at 115 mg/kg/day (not always statistically significant). Based on the minimal magnitude of the change and based on the direction of the change this was considered not toxicologically relevant.
Body weights and body weight gain of treated animals up to 60 mg/kg/day remained in the same range as controls over the treatment period.
Any other statistically significant changes in body weight (gain) were considered not related to treatment with the test item as those occurred in absence of a dose-response relationship. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- A slightly increased food consumption was noted for females treated at 115 mg/kg/day during (part of) the pre-mating period. Based on the magnitude of the change and as this did not continue during the remainder of the study, this was considered not toxicologically relevant.
The higher relative food consumption noted for males treated at 115 mg/kg/day was considered to be caused by the lower body weights.
Further statistically significant changes in food consumption before or after correction for body weight were considered not toxicologically relevant since no trend was apparent regarding dose and duration of treatment. Note: When compared to the pre-mating period, a generally higher food intake (absolute and relative to body weight) was measured in females during the phases of gestation and lactation, with values for the control group within the normal range. The observed higher food intake is a normal physiological process. It reflects the increased nutritional need of the dams during the periods of gestation and lactation. Furthermore, from the third week of lactation onwards also their pups start to consume solid feed. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles. - Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related changes were noted in white blood cell parameters in males at 115 mg/kg/day: increased concentrations of white blood cells (WBC; 1.66x of control), mainly caused by an increased concentration of neutrophils and lymphocytes (2.56x and 1.45x, respectively). In addition, changes in platelets and red blood cell parameters were noted: increased concentrations of platelets and reticulocytes (1.42x and 1.32x, respectively), increased red cell distribution width (RDW; 1.08x), and decreased mean corpuscular volume and mean corpuscular haemoglobin (MCV and MCH; 0.96x and 0.95x, respectively).
For females at 115 mg/kg/day, only RDW was increased (1.10x). The decreased haemoglobin concentration in males at 115 mg/kg/day (0.95x) might have
been the result of slightly high control values.
It should be noted that high individual values were noted for Male Nos. 87, 89, 90, 91 and/or 93 at 115 mg/kg/day for WBC, lymphocytes, monocytes, reticulocytes and/or platelets.
No test item-related changes were noted in hematological parameters in animals treated up to 60 mg/kg/day.
Other statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in absence of a dose-related trend. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related changes were noted in males at 115 mg/kg/day: increased alanine aminotransferase (ALAT; 3.63x of control), aspartate aminotransferase (ASAT; 2.92x) and alkaline phosphatase (ALP; 1.63x) activities, and increased total bilirubin (1.81x) and bile acids (4.00x) concentrations. These changes were mainly caused by high values in up to 8/10 animals; extreme high values were noted for Male Nos. 86, 90 and 93. In addition, for Male No. 90, (extreme) low values were noted for total protein, albumin, urea and potassium. For females at 115 mg/kg/day, high ALAT and ASAT activities, and high bilirubin concentrations were noted in only 1/9 animals.
Other statistically significant changes in clinical biochemistry parameters were considered not toxicologically relevant based on the magnitude of the change and/or absence of a dose response relationship.
Serum levels of TSH and T4 in F0-males and -females were considered not to be affected by treatment with the test item.
The statistically higher TSH concentration in males at 25 mg/kg/day was considered to be unrelated to treatment with the test item as this occurred in the absence of a dose-related trend. - Endocrine findings:
- not specified
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- 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 were noted in the liver of the 60 and 115 mg/kg/day group males and females and in the spleen of the males treated at 60 and 115 mg/kg/day.
Liver: A combination of many liver findings within the periportal areas were observed in males and females treated at 60 and 115 mg/kg/day and consisted of:
- Basophilia and/or karyocytomegaly in males starting at 60 mg/kg/day and in females at 115 mg/kg/day up to moderate degree.
- Peribiliary infiltration in males starting at 60 mg/kg/day up to moderate degree and in females at 115 mg/kg/day up to slight degree.
- Periportal fibrosis, in most cases with the presence of pigmented macrophages, in males starting at 60 mg/kg/day and in females at 115 mg/kg/day up to moderate degree.
- Periportal necrosis in males starting at 60 mg/kg/day up to marked degree and in females at 115 mg/kg/day up to moderate degree
- Bile duct hyperplasia in males starting at 60 mg/kg/day up to moderate degree and in females at 115 mg/kg/day up to slight degree.
The liver findings (based on necrosis and fibrosis) in males at 60 and 115 mg/kg/day and in females at 115 mg/kg/day were considered adverse.
Spleen: An increased incidence and severity of extramedullary hematopoiesis up to moderate degree in males starting at 60 mg/kg/day. The spleen findings were considered non-adverse.
The presence of minimal to slight extramedullary hematopoiesis in the spleen is frequently observed in control animals as a normal background finding. Therefore, the few treated females with minimal to slight extramedullary hematopoiesis in the spleen were considered not related to the test-item.
In the bone marrow of treated females, the incidence and severity of adipose tissue seemed to be slightly increased. However, based on the lack of a dose response relationship this finding was considered not test item-related.
Remaining histologic changes were considered to be incidental findings or 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. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- No toxicologically relevant changes in coagulation parameters were noted.
A slightly shorter (not statistically significant) activated partial thromboplastin time (APTT) was noted for females treated at 115 mg/kg/day, this was considered not to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity. - 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.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for Female No. 108 of the control group and Female No. 183 at 115 mg/kg/day (both delivered a litter). Given their incidental nature and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment with the test item. - Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- Sperm motility, morphology and concentration were considered unaffected by treatment with the test item.
Any statistically significant changes in sperm analysis were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend. - Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Mating Index: Mating index was considered not to be affected by treatment with the test item.
All females showed evidence of mating. The mating indices for males were 100% for the control, 25 and 60 mg/kg/day groups and 96% for the 115 mg/kg/day group.
Male No. 83, treated at 115 mg/kg/day, did not mate successfully as this animal was euthanized for humane reasons on Day 6 of the mating period. Female No. 183, which was initially cohabitated with Male No. 83, showed evidence of mating after it was cohabitated with Male No. 81.
Precoital Time: Precoital time was considered not to be affected by treatment with the test item.
All females showed evidence of mating within 4 days, except for one animal of the control group and one animal treated at 115 mg/kg/day, which showed evidence of mating after 12 days (Nos. 103 and 200). Based on the incidence this was considered unrelated to treatment with the test item.
Female No. 183 showed evidence of mating 1 day after she was cohabitated with Male No. 81
Number of Implantation Sites: Number of implantation sites was considered not to be affected by treatment.
Female Nos. 108 (control group) and 156 (60 mg/kg/day) had only 3 and 1 implantation sites, respectively. As this occurred incidentally and in absence of a dose response relationship, this was considered to be unrelated to treatment with the test item.
Fertility Index: Fertility index was considered not to be affected by treatment.
The fertility indices for females were 96, 96, 100 and 100% for the control, 25, 60 and 115 mg/kg/day groups, respectively. The fertility indices for males were 96, 96, 104 and 100% for the control, 25, 60 and 115 mg/kg/day groups, respectively. One female (No. 124) of the control group and one female (No. 140) at 25 mg/kg/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. The fertility index of 104% of males at 60 mg/kg/day can be explained by that Animal No. 63 was mated with Animal Nos. 162 and 163 as Animal No. 62 was sacrificed in extremis before the initiation of mating.
Histopathological Evaluation of Reproductive Performance: In the control group and the 25 mg/kg/day treatment group there were 1/25 couples not pregnant (Female No. 124 / Male No. 24; Female No. 140 / Male No. 40) , there was 1/25 females at 60 mg/kg/day with total litter loss (Female No. 156) and 1/25 females with implantation sites only (Female No. 199).
Except for the presence of dead pups in the uterus of Female No. 199 which correlates with the presence of implantation sites only, no abnormalities were seen in the reproductive organs or mammary gland of the other couples or female, that could explain the absence of pregnancy and total litter loss.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Gestation Index and Duration: Gestation index and duration of gestation were considered not to be affected by treatment.
Except for one female (No. 199) at 115 mg/kg/day, all pregnant females had live offspring. The gestation indices were 100, 100, 100 and 96% for the control, 25, 60 and 115 mg/kg/day groups, respectively.
Female No. 199 was sacrificed in extremis on post-coitum Day 21. This failed pregnancy was without related histopathology changes in reproductive organs. Due to the incidental occurrence, the gestation index was judged to be unaffected by treatment with the test item.
Parturition/Maternal Care: Female No. 182 (treated at 115 mg/kg/day) was sacrificed in extremis on lactation Day 1, she did not clean herself nor the pups after given birth and no/only a little milk was seen in the stomach of the pups. This was considered to be related to maternal toxicity, rather than an effect of treatment with the test item on maternal care.
No signs of difficult or prolonged parturition were noted among the pregnant females and examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Post-Implantation Survival Index: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 95, 96, 94 and 92% for the control, 25, 60 and 115 mg/kg/day groups, respectively.
For one female at 25 mg/kg/day (No. 136), the number of pups was slightly higher than the number of implantations (11 implantations vs. 12 pups). This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 21-23 days of lactation. No toxicological relevance was attached to this finding in the current study.
Litter Size: Litter size was considered not affected by treatment.
Mean litter sizes were 11.3, 11.3, 11.1 and 12.1 living pups/litter for the control, 25, 60 and 115 mg/kg/day groups, respectively.
Sex Ratio- F1 Pups: Sex ratio was considered not to be affected by treatment
Live Birth Index: Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be affected by treatment. The live birth indices were 100, 100, 99 and 100% for the control, 25, 60 and 115 mg/kg/day groups, respectively.
One pup of the control group, 4 pups at 60 mg/kg/day and 1 pup at 115 mg/kg/day 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.
Viability Index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered to be unaffected by treatment.
Viability indices (the number of live offspring on Day 4 before culling compared to the number of offspring on Day 1) were 99, 100, 100 and 95% for the control, 25, 60 and 115 mg/kg/day groups, respectively.
Three pups of the control group, one pup at 25 mg/kg/day, one pup at 60 mg/kg/day and three pups at 115 mg/kg/day were found dead or missing between PND 2 and 4. 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.
In addition, all thirteen pups of Dam No. 182 at 115 mg/kg/day were sacrificed on PND 1 as the dam was sacrificed in extremis (for details see Section 9.2.1). Body weight of these pups were lower, when compared to those of other litters on PND 1. This was considered to be related to maternal toxicity.
Weaning Index: The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment.
The weaning indices were 99, 98, 100 and 99% for the control, 25, 60 and 115 mg/kg/day groups, respectively.
One pup of the control group, 3 pups at 25 mg/kg/day and 1 pup at 115 mg/kg/day were found dead or missing between culling (PND 4) and weaning (PND 21). Pups missing were most likely cannibalized. 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. - Dose descriptor:
- NOAEL
- Remarks:
- Development/Reproduction
- Effect level:
- >= 115 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other:
- Remarks on result:
- other: No adverse effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- General toxicity
- Effect level:
- 25 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- mortality
- other: adverse liver findings starting at 60 mg/kg/day
- Critical effects observed:
- not specified
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No clinical signs occurred among pups that were considered to be related to treatment. Paralyzed hindlegs were noted for one pup of the control group, one pup at 25 mg/kg/day, 3 pups at 60 mg/kg/day and 1 pup at 115 mg/kg/day. In absence of a dose-related trend this was considered unrelated to treatment.
The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Data (Cohort 1A and 1B) from weaning onwards: No toxicologically relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during weekly arena observations.
Salivation seen after dosing among animals treated at 60 and 115 mg/kg/day during the treatment period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item and vehicle rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period 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. - Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- Data (Cohort 1A and 1B) from weaning onwards: No mortality occurred during the study period.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No toxicologically relevant change in body weights of pups was observed.
Slightly lower body weights were noted for pups at 115 mg/kg/day (0.96x of control at PND 21 for males and females combined; not statistically significant). Based on the magnitude of the change this was considered not toxicologically relevant.
Data (Cohort 1A and 1B) from weaning onwards: No toxicologically relevant changes were noted in body weight of body weight gain.
A slightly reduced body weight gain was noted for males at 115 mg/kg/day, resulting in body weights of 0.96x of control at the end of treatment (not statistically significant). Based on the magnitude of the change this was considered not toxicologically relevant.
Any statistically significant changes in body weight or body weight gain were considered unrelated to treatment with the test item since no trend was apparent regarding dose and/or duration of treatment. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Data (Cohort 1A and 1B) from weaning onwards: Food consumption before or after correction for body weight was similar to the control level over the study period.
Any statistically significant changes in food consumption before or after correction for body weight were considered unrelated to treatment with the test item since no trend was apparent regarding dose and/or duration of treatment. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related increased white blood cells were noted in males at 115 mg/kg/day (WBC; 1.37x of control), which was mainly caused by an increased concentration of neutrophils and lymphocytes (1.64x and 1.34x, respectively, not statistically significant). In addition, increased concentrations of platelets and reticulocytes (1.28x and 1.21x, respectively; not statistically significant for reticulocytes) and decreased mean corpuscular haemoglobin (MCH; 0.95x) were noted for males at 115 mg/kg/day.
For females at 115 mg/kg/day, increased red blood cell concentration and haematocrit (1.09x for both parameters) were noted.
Other statistically significant changes in hematology parameters were considered to be unrelated to treatment with the test item as these occurred in absence of a dose-related trend.
No toxicologically relevant changes in coagulation parameters were noted.
A shorter activated partial thromboplastin time (APTT) was noted for females treated at 115 mg/kg/day, this was considered not to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
It should be noted that high individual values of the parameters above were noted for Male Nos. 443, 445, 449, 454 and/or 455.
No test item-related changes were noted in hematological parameters in animals treated up to 60 mg/kg/day. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Serum T4 levels in male and female pups, culled at PND 4 and male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment.
Serum T4 and TSH levels in male and female pups of Cohort Surplus at PND 22 were considered not to be affected by treatment.
Note to table: Serum T4 levels of PND 4 pups are reported under F0-females, as these were derived from pooled samples collected from male and female pups.
Cohort 1A
Test item-related changes (not always statistically significant) were noted in males and females at 115 mg/kg/day: increased alanine aminotransferase (ALAT; 4.47x and 3.52x of control, respectively), aspartate aminotransferase (ASAT; 3.26x and 2.36x) and alkaline phosphatase activities (ALP; 1.47x and 1.41x), and urea (1.20x and 1.11x), bile acid (2.12x and 1.50x) and inorganic phosphate concentrations (1.24x and 1.19x).
It should be noted that high individual values of (most of) the parameters above were noted for Male Nos. 443, 444, 449, 453 and/or 459 and Female Nos. 764, 778, 779 and/or 780.
Other statistically significant changes in clinical biochemistry parameters were considered not toxicologically relevant based on the magnitude of the change and/or absence of a dose response relationship.
Thyroid hormone analyses: An increased concentration of TSH was noted for males and females at 115 mg/kg/day (2.81x and 1.86x of control, respectively; not statistically significant). - Urinalysis findings:
- not examined
- Sexual maturation:
- no effects observed
- Description (incidence and severity):
- Balanopreputial separation: For males at 115 mg/kg/day, mean time to balanopreputial separation was increased (43.8 days vs. 42.4 days for control). Body weights of animals on the day balanopreputial separation was observed was similar to controls and mean values remained within the historical control data.
For females at 115 mg/kg/day, the time of onset until a vaginal opening (33.2 vs. 30.7 days for control) and the time until the first estrus (5.3 vs. 4.2 days, not statistically significant) were increased. These periods remained within the range of historical control data.
Cohort 1A
Estrous Cycle: An irregular estrous cycle was noted for Females Nos. 683 and 693 at 60 mg/kg/day, and for Female Nos. 771, 774 and 777 and 780 at 115 mg/kg/day. At 115 mg/kg/day, regularity of the estrous cycle could not be determined for Female Nos. 769 and 775, and Female No. 766 had an extended estrus cycle. No treatment related effect on the estrous cycle was observed for females treated at 25 mg/kg/day. - 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 115 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- No test item-related effects on organ weights were noted for Cohort Surplus pups.
Any statistically significant changes in organ weights of Cohort Surplus pups were considered to be unrelated to treatment with the test item as these occurred in absence of a dose-related trend.
Cohort 1A and 1B (PND 89-95)
Test item-related higher liver and spleen weights were noted in males treated at 115 mg/kg/day.
In females the increased liver weight was very small, only limited to the relative weight and the same magnitude as the increase in liver weight in males at 60 mg/kg/day which was not statistically significant, suggesting that a test item-relationship is questionable. However, based on the many macroscopic and microscopic findings, a test-item relationship cannot fully be excluded.
Some organ weight differences in males were statistically significant when compared to the control group (absolute weight: prostate; relative weight: brain, testes) but were considered to be the result of a test item-related effect on final body weight.
The statistically significant lower adrenal gland weights in females were considered not to be test item-related due to the magnitude of the changes, the direction of the change (lower weight), the absence of any macroscopic or microscopic findings and the fact that this change was not observed in the females of cohort 1B (approximately the same age and the same group size) and values were all within the adrenal gland weight values of historical control data.
There were no other test item-related organ weight changes.
Cohort Surplus (PND 22)
Brain, thymus and spleen weight were considered not affected by treatment with the test item. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment.
Cohort 1A (PND 89-95)
Test item-related macroscopic findings were noted in the liver of males and females starting at 60 mg/kg/day and in the spleen of males and females at 115 mg/kg/day, and consisted of:
Liver:
- Yellowish/reddish focus/foci in 1/20 females at 25 and 60 mg/kg/day and in 11/20 males and 3/20 females at 115 mg/kg/day. The microscopic correlate was periportal necrosis or the presence of pigmented macrophages (in fibrotic areas).
- Accentuated lobular pattern in 3/20 males and 1/20 females at 115 mg/kg/day. The microscopic correlate was periportal fibrosis.
- Enlarged in 1/20 males at 25 and 60 mg/kg/day and in 3/20 males at 115 mg/kg/day; thickened in 2/20 males and 1/20 females at 115 mg/kg/day. There was no clear microscopic correlate.
- Reduced in size in 2/20 males and females at 115 mg/kg/day. The microscopic correlate was periportal fibrosis.
- Grown together with adjacent tissue in 2/20 males at 115 mg/kg/day.
Spleen:
- Enlarged in two males and a single female at 115 mg/kg/day. The microscopic correlate was moderate extra-medullary hematopoiesis. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Cohort 1B (≥ PND 90)
Test item-related macroscopic findings were noted in the liver of males starting at 60 mg/kg/day, and consisted of:
Liver:
- Yellowish/reddish focus/foci in 2/20 and 6/20 males at 60 and 115 mg/kg/day, respectively.
- Accentuated lobular pattern in 2/20 males at 115 mg/kg/day.
- Enlarged in 1/20 males at 25 and 60 mg/kg/day, and in 5/20 males at 115 mg/kg/day; and thickened in 1/20 males and 2/20 males at 60 and 115 mg/kg/day, respectively.
- Reduced in size in 1/20 and 2/20 males at 60 and 115 mg/kg/day, respectively.
Grown together with adjacent tissue in 1/20 and 2/20 males at 60 and 115 mg/kg/day, respectively.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Cohort Surplus (PND 22)
No test item-related macroscopic findings were noted. - Histopathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings were noted in the liver of males at 115 mg/kg/day and of females starting at 25 mg/kg/day, and in the spleen of the males starting at 60 and females at 115 mg/kg/day.
These findings are summarized in the text table below.
Liver:
A combination of many liver findings within the periportal areas were observed in males and females treated at 60 and 115 mg/kg/day and consisted of:
- Basophilia and/or karyocytomegaly at 115 mg/kg/day in males up to moderate degree and in females up to slight degree.
- Peribiliary infiltration in males at 115 mg/kg/day up to moderate degree and in females staring at 25 mg/kg/day up to slight degree.
- Periportal fibrosis in males at 115 mg/kg/day up to marked degree and in females starting at 25 mg/kg/day up to moderate degree.
- Periportal pigmented macrophages at 115 mg/kg/day in males up to moderate degree and in females at minimal degree.
- Periportal necrosis at 115 mg/kg/day in males up to marked degree and in females at up to moderate degree
- Bile duct hyperplasia at 115 mg/kg/day in males up to marked degree and in females at up to moderate degree.
The liver findings (based on necrosis and fibrosis) in males 115 mg/kg/day and in females starting at 25 mg/kg/day were considered adverse.
Spleen:
An increased severity of extramedullary hematopoiesis up to moderate degree in males starting at 60 mg/kg/day and in females at 115 mg/kg/day. The spleen findings were considered non-adverse.
The presence of minimal to slight extramedullary hematopoiesis in the spleen is regularly observed in control animals and therefore considered to be within background. Remaining histologic changes were considered to be incidental findings or 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.
Ovarian Follicle Counts (Cohort 1A)
There were no test item-related effects on the ovarian follicle counts in the F1-group females (Cohort 1A) treated at 115 mg/kg/day when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant. - Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Cohort 1A
Sperm Analysis: A reduced number of sperm cells were counted for males at 115 mg/kg/day (0.75x of control). These findings were noted at the high dose in which also systemic toxicity was noted including three F0-mortalities.
No test item-related changes were noted in motility or morphology of the sperm cells. - Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- LOAEL
- Remarks:
- General toxicity
- Generation:
- F1
- Effect level:
- 25 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- histopathology: non-neoplastic
- Dose descriptor:
- NOAEL
- Remarks:
- Developmental
- Generation:
- F1
- Effect level:
- >= 115 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other:
- Remarks on result:
- other: No adverse effects observed
- Dose descriptor:
- NOAEL
- Remarks:
- Reproduction
- Generation:
- F1
- Effect level:
- 60 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other:
- Remarks on result:
- other: These findings were noted at the high dose in which also systemic toxicity was noted including three F0-mortalities.
- Critical effects observed:
- not specified
- Reproductive effects observed:
- not specified
- Conclusions:
- In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohort 1), the following No Observed Adverse Effect Level (NOAELs) of Diallyl 2,2’-oxydiethyl dicarbonate were established:
NOAEL (general toxicity, F0) 25 mg/kg/day, based on test item related premature sacrifices at 115 mg/kg/day and adverse liver findings starting at 60 mg/kg/day.
NOAEL (general toxicity, F1) could not be established as adverse findings were noted in the liver starting at 25 mg/kg/day.
NOAEL (reproduction, F0) >=115 mg/kg/day (no reproduction toxicity was observed up to the highest dose level tested).
NOAEL (reproduction, F1) 60 mg/kg/day (based on irregular estrous cycles and lower
sperm counts; findings were noted at the high dose in which also systemic toxicity was noted including three F0-mortalities).
NOAEL (development) >= 115 mg/kg/day (no developmental toxicity was observed up to the highest dose level tested). - Executive summary:
The Extended One-Generation Reproductive Toxicity Study was conducted according to OECD 443 (adopted June 2018) and in compliance with GLP. The objective of this study was to provide an evaluation of the pre- and postnatal effects of Diallyl 2,2’-oxydiethyl dicarbonate on reproduction and development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring.
In addition, the study provided and/or confirmed information about the effects of the test item on the integrity and performance of the adult male and female reproductive systems. Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation.
The dose levels in this study were selected to be 0, 25, 60 and 115 mg/kg/day, based on the results of an oral 90-day repeated dose toxicity study and an oral Reproduction/Developmental Toxicity Screening Test in an attempt to produce graded responses to the test item.
Chemical analyses of formulations were conducted at three occasions during the study to assess accuracy and homogeneity.
For the F0-generation, the following parameters and end points were evaluated: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations.
For the F1-generation, the following parameters and end points were evaluated: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, clinical pathology including measurement of thyroid hormones, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations.
In addition, the following reproduction/developmental parameters were determined: estrous cycle determination, 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, macroscopy and measurement of thyroid hormones).
Test formulations prepared were considered accurate and homogenous. For the formulation of Group 3 and Group 4 prepared for use in Week 11, the mean accuracy was slightly below the target concentration (i.e. 87% of target). As formulation prepared for Weeks 1 and 22 were within specifications, and as the deviation from the acceptance criteria was small, the formulations were considered acceptable.
F0-generation - Parental results
A total of four animals were sacrificed prematurely. At 115 mg/kg/day, one male and two females were sacrificed in extremis on Days 77-95 based on their clinical condition and/or body weight loss. Correlating treatment related macroscopic and/or microscopic findings were noted in the liver, thymus and/or kidneys. At 60 mg/kg/day, one male was sacrificed in extremis on Day 66 based on its clinical condition. Macroscopically, grey-white foci were noted on the lungs, and misdosing was suspected. No microscopic findings were noted that could explain the moribund condition.
Microscopic findings of surviving animals were noted in the liver and spleen. Liver findings in males started at 60 mg/kg/day, and in females at 115 mg/kg/day. Findings were mainly concentrated in the periportal areas of the liver and represented a cascade of events related to hepatocellular toxicity. This cascade most likely started with test item-induced hepatocellular changes (basophilia and karyocytomegaly) and peribiliary infiltration of mononuclear cells followed by hepatocellular necrosis and a regeneration process expressed by presence of pigmented macrophages, fibrosis and bile duct hyperplasia. These findings correlated with increased liver enzyme activities and bile acids in males at 115 mg/kg/day. Particularly necrosis is degenerative in nature and therefore considered adverse, but also fibrosis may significantly affect the normal structure and function of the liver and is therefore also considered adverse.
In the spleen, an increased incidence and/or severity of extramedullary hematopoiesis in males starting at 60 mg/kg/day was observed, which could be related to changes in blood cell parameters (mainly increased white blood cell parameters, reticulocytes and platelets in males at 115 mg/kg/day). These changes were considered to be indirectly related to the liver findings due to a reaction on the necrosis and/or inflammation in the liver. Based on the relative low magnitude of the extramedullary hematopoiesis (compared to controls and background severities), these changes were considered non-adverse.
Clinical pathology changes a 115 mg/kg/day that were also noted, included decreased mean corpuscular volume and mean corpuscular haemoglobin and increase bilirubin concentrations (males) and increased red cell distribution width (both sexes). In absence of correlating microscopic findings these changes were considered non adverse.
Body weight (gain) in males at 115 mg/kg/day from Week 5 onwards was decreased. Based on the magnitude of the change this was considered non-adverse.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, food consumption, coagulation parameters and thyroid hormone levels).
F0-generation-Reproductive results
No reproduction toxicity was observed up to the highest dose level tested (115 mg/kg/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).
F0-generation-Developmental results
No developmental toxicity was observed up to the highest dose level tested (115 mg/kg/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 and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels and macroscopic examination).
F1-generation results
Microscopic findings were noted in the liver and spleen, which were comparable to the
effects noted in the F0-generation.
Liver findings were noted in males and females at 115 mg/kg/day and in single females at 60 and 25 mg/kg/day. Findings were mainly concentrated in the periportal areas of the liver and represented a cascade of events related to hepatocellular toxicity. This cascade most likely started with test item-induced hepatocellular changes (basophilia and karyocytomegaly) and peribiliary infiltration of mononuclear cells followed by hepatocellular necrosis and a regeneration process expressed by presence of pigmented macrophages, fibrosis and bile duct hyperplasia. These findings correlated with increased clinical pathology liver parameters (liver enzymes and/or bile acids) observed at 115 mg/kg/day. Particularly necrosis is degenerative in nature and therefore considered adverse, but also fibrosis may significantly affect the normal structure and function of the liver and is therefore also considered adverse.
As the findings in the single females at 60 and 25 mg/kg/day were similar compared to the findings in multiple animals at 115 mg/kg/day, these findings were considered adverse in females starting at 25 mg/kg/day.
In the spleen, an increased incidence and/or severity of extramedullary hematopoiesis in males starting at 60 mg/kg/day and females at 115 mg/kg/day, which could be related to changes in blood cell parameters (mainly increased white blood cells, reticulocytes and platelets in males at 115 mg/kg/day). These changes were considered to be indirectly related to the liver findings due to a reaction on the necrosis and/or inflammation in the liver. Based on the relative low magnitude of the extramedullary hematopoiesis (compared to controls and background severities), these changes were considered non-adverse.
Clinical pathology changes at 115 mg/kg/day that were also noted included increased urea, inorganic phosphate and TSH concentrations (males and females), increased red blood cell concentrations and haematocrit (females), and decreased mean corpuscular haemoglobin (males). In the absence of correlating microscopic findings these changes were considered non-adverse.
The number of sperm cells was reduced in males at 115 mg/kg/day, while all other sperm parameters were unaffected. In females at 115 mg/kg/day estrus was affected, consisting of irregular (4/20 females), extended (1/20) and undetermined regularity (2/20). An irregular estrous cycle was also noted at 60 mg/kg/day (2/20). The effects on sperm cells and estrous cycle at 115 mg/kg/day were considered adverse based on the magnitude of the change or the incidence. These findings were noted at the high dose in which also systemic toxicity was noted including three F0-mortalities.
Sexual maturation at 115 mg/kg/day was slightly delayed for both sexes. The time to balanopreputial separation, and onset until vaginal opening and first estrus were increased. For males, body weights of the animals at balanopreputial separation were similar to control. Therefore, this delay was considered related to a slight delay in development and was considered not toxicologically relevant. For females, body weights at vaginal opening and first estrus were higher compared to control and a relation to treatment with the test item could not be excluded. However, as changes were minimal and as the periods remained within the historical control data, this was considered non-adverse.
The results of other endocrine disruptor measurements evaluated (thyroid hormone measurements, nipple retention, anogenital distance, and histopathology of endocrine organs) were considered not be affected by treatment with the test item.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, body weights, food consumption, coagulation parameters, splenic lymphocyte subpopulation and ovarian follicle counts).
In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohort 1), the following No Observed Adverse Effect Level (NOAELs) of Diallyl 2,2’-oxydiethyl dicarbonate were established:
NOAEL (general toxicity, F0) 25 mg/kg/day, based on test item related premature sacrifices at 115 mg/kg/day and adverse liver findings starting at 60 mg/kg/day.
NOAEL (general toxicity, F1) could not be established as adverse findings were noted in the liver starting at 25 mg/kg/day
NOAEL (reproduction, F0) >=115 mg/kg/day (no reproduction toxicity was observed up to the highest dose level tested)
NOAEL (reproduction, F1) 60 mg/kg/day (based on irregular estrous cycles and lower sperm counts; findings were noted at the high dose in which also systemic toxicity was noted including three F0-mortalities)
NOAEL (development) >= 115 mg/kg/day (no developmental toxicity was observed up to the highest dose level tested)
Referenceopen allclose all
No test article-related macroscopic changes were observed in rats of either sex in either the repeated dose component or the reproductive component of the study that were treated with ADC.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effect of treatment with ADC was evident from body weight gain for females in the reproductive component during 2-wk premating period, gestation and over Lactation Days 0 to 4. Mean body weight gain during these periods for the treated groups were comparable to control group.
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not applicable (dermal study)
REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
not performed
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
not performed
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effect of treatment with ADC was evident from reproductive performance indices. Fertility and fecundity indices ranged between 90 to 100% in the treated groups and were comparable to the 100% in controls. The mean number of days-to-mating for the treated groups ranged from 3.2 to 4.4 days and was considered comparable to the 2.1 days in the controls. Mating indices were 100% in the control and each treated group.
No effect of treatment with ADC was evident from parturition data. The number of females that delivered litters with at least one viable pup in the control, 150, 454, and 1030 mg/kg/day groups was 10, 10, 9, and 10, respectively. The Gestation Index was 100% in the control and each treated group. Mean gestation length in the treated groups ranged from 22.1 to 22.4 days and was comparable to the 22.1 days in controls. The mean number of total (live plus dead), liveborn, and stillborn pups per litter in the treated groups was comparable to controls. Likewise, the Live Birth and Stillborn Indices for the treated groups were comparable to controls. The mean number of uterine implantation scars/female determined at terminal necropsy for the treated groups was comparable to controls and in all groups and corresponded closely with the mean total number of pups (live plus dead) at birth.
ORGAN WEIGHTS (PARENTAL ANIMALS)
No test article-related organ weight changes were noted in rats of either sex that received ADC in the repeat dose or reproductive phase of the study. Absolute mean spleen weight and relative spleen weight to body and brain weights were statistically significantly decreased in female rats (reproductive component) that received 150 mg/kg/day ADC compared to controls, but the decrease was not dose related and there were no corresponding microscopic changes in spleens of repeat dose animals. The spleen weight decrease was considered to be spurious.
GROSS PATHOLOGY (PARENTAL ANIMALS)
No test article-related macroscopic changes were observed in rats of either sex that were treated with ADC.
HISTOPATHOLOGY (PARENTAL ANIMALS)
No test article-related microscopic changes were noted in male rats that received repeated doses of ADC. Microscopic changes observed were typical of those commonly found in animals of the same strain and age and were considered to be incidental
OTHER FINDINGS (PARENTAL ANIMALS)
NEUROPATHOLOGICAL EVALUATIONS
In the neuropathological evaluations of selected nervous tissues conducted by EPLS, minimal axonal degeneration characterized by either the presence of digestion chambers and /or swollen axons (spheroids) was observed in one level of spinal cord from one high-dose (1030 mg/kg/day) female and from one mid-dose (454 mg/kg/day) female. The sciatic nerve of one high-dose female and two mid-dose females also had minimal axonal degeneration. Similar minimal changes were not observed in the control animals. These minimal lesions were considered well within the range of what could occur as an incidental background finding. Thus, it was concluded that since there was no dose response, the changes seen were probably incidental background findings unrelated to test article treatment.
No effect of treatment with ADC was evident from Fi pup survival to PND 4. Pup survival indices over PND 0-4 (i.e. Viability Index) in the treated groups ranged from 88.62 to 98.71% and were comparable to the 98.12% in controls. The relatively low Viability Index in the 150 mg/kg/day group (88.62%) was attributed to the death of the one Iivebom pup in the litter of Female Number 293. This female delivered a litter of two pups. One pup was alive but died soon after birth, and the other pup was partially cannibalized when found after delivery. At necropsy, this female only had two implantation scars.
CLINICAL SIGNS (OFFSPRING)
No effect of treatment with ADC was evident from Fi pup clinical examinations at birth or at Postnatal Day (PND) 4. The few findings seen among the treated pups occurred with low incidence, and were considered spurious and unrelated to treatment.
BODY WEIGHT (OFFSPRING)
No effect of treatment with ADC was evident from Fi pup body weights. These weights distinguished by sex and for both sexes combined in the treated groups were comparable to controls at birth and PND 4.
SEXUAL MATURATION (OFFSPRING)
Not applicable
ORGAN WEIGHTS (OFFSPRING)
Not exmined
GROSS PATHOLOGY (OFFSPRING)
No effect of treatment with ADC was evident from the external examination of Fi stillborn pups, pups that died on study, or pups euthanized on PND 4. No findings were seen among the treated or control pups.
HISTOPATHOLOGY (OFFSPRING)
not examined
OTHER FINDINGS (OFFSPRING)
SEX RATIO:
No effect of treatment with ADC was evident from F] pup sex ratios. Mean pup sex ratios at birth for the treated groups ranged from 46.39 to 50.62% and were considered comparable to the 55.38% in controls. Consistent with the good pup survival in all groups, these sex ratios at PND 4 changed little from those at birth.
Results F0
Mean Percent Liver, Spleen and Thymus Weight Differences From Control Groups - F0-Generation
|
Males |
Females |
||||
Dose level (mg/kg/day): |
25 |
60 |
115 |
25 |
60 |
115 |
LIVER |
|
|
|
|
|
|
Absolute |
3 |
10 |
14** |
-1 |
4 |
8* |
Relative to body weight |
6 |
8 |
27** |
-2 |
2 |
4 |
SPLEEN |
|
|
|
|
|
|
Absolute |
-5 |
2 |
7 |
2 |
6 |
11* |
Relative to body weight |
-3 |
1 |
19** |
1 |
4 |
7 |
THYMUS |
|
|
|
|
|
|
Absolute |
-3 |
-2 |
-14 |
-13 |
-10 |
-15* |
Relative to body weight |
1 |
-3 |
-5 |
-12 |
-11 |
-18** |
*: P<0.05, **: P<0.01
Further results (F1) Cohort 1A
Splenic Lymphocyte Subpopulation: There were no test item-related effects on splenic lymphocyte subpopulations observed. A slightly lower T-cytotoxic cell splenic subpopulation was observed for females at 60 mg/kg/day. As this shift was slight of nature and occurred in the absence of a dose-related trend, this was considered to represent biological variability and considered not to be related to treatment.
Effect on fertility: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 60 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Data are of good quality (OECD Guideline Tests conduced in compliance with GLP)
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 030 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
Additional information
The Combined Repeat Dose Toxicity Study with a Reproductive/Developmental screening Test comprises of two components, a repeat dose toxicity study with neurobehavioral evaluations and a reproduction/developmental toxicity screening (refer also to section 7.5.3 Repeated dose toxicity: dermal).
The purpose of the reproduction/developmental toxicity screening component was to provide information on possible effects on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus, and parturition. Both the repeat dose and the reproductive/developmental component were comprised of three treatment groups and a saline-treated control group. Each reproductive group contained ten female Sprague-Dawley rats) [Crl: CD" (SD)IGS BR]. The ten male animals of each repeat dose group were also utilized for the reproductive/developmental component of the study. Animals were administered dermally once daily via occlusion for 6 hours at dose levels 150, 454, and 1030 mg/kg/day for at least 42 consecutive days, while females in the reproductive component were treated for two weeks before pairing, during pairing, and from Gestation Days (GD) 0 to 20. The control animals received 0.9% Sodium Chloride, USP, at a volume of 0.9 mL/kg for the same duration as the treated animals. After two weeks of treatment, the animals were cohabited nightly with males from the repeat dose component, one male to one female, from the same treatment group, for up to 14 days. Females were evaluated daily for evidence of mating (sperm in the vaginal rinse or vaginal plug). Once mating was confirmed (GD 0), females were separated from the male for the remainder of gestation, and allowed to deliver and nurse litters until Postnatal Day (PND) 4. Litter size and pup evaluations (body weight, sexing, and external examination) were recorded at birth and PND 4. Pups were euthanized and externally examined on PND 4 and the carcasses were discarded without further examination. Complete necropsies were performed on all parent animals (repeat dose and reproductive components) and organs and tissues were collected, weighed, and preserved.
No effect of treatment was evident from mortality, clinical evaluations, dermal evaluations, body weights, food consumption, organ weights, macroscopic or microscopic evaluations, reproductive performance, gestation and lactation body weights or food consumption, gestation length, litter size, pup body weight, pup sex ratios, or pup external examinations to PND 4. Thus, in this rat dermal repeat dose toxicity study with a reproduction/developmental toxicity component, the No-Observable-Adverse-Effect Level (NOAEL) of the test article diallyl diglycol carbonate, for parental toxicity was 1030 mg/kg/day, the highest dose level evaluated. The No-Observable-Effect Level (NOEL) of reproductive performance was 1030 mg/kg/day, the highest dose level evaluated.
A teratology study (Bio-Research Laboratories Ltd, 1985) provides also sufficient information on effects of diallyl diglycol carbonate on reproductive performance and fertility. In this study, New Zealand white rabbits were exposed dermally to extremely high doses (114, 572 and 1143 mg/kg bw/day) that resulted in severe skin lesions (described as blackened thickened skin) in the dams of all exposure groups. Marked body weight losses and abortion were observed for the mid and high dose groups and 7/18 dams in the high dose group died. An increased incidence of resorptions and ocular lesions and a decrease in incidence of single thirteenth ribs was noted for the mid dose group. However, due to the excessive maternal toxicity noted at these dose levels, the reproductive and developmental effects noted in this study are not considered to be relevant.
The Extended One-Generation Reproductive Toxicity Study was conducted according to OECD 443 (adopted June 2018) and in compliance with GLP. The objective of this study was to provide an evaluation of the pre- and postnatal effects of Diallyl 2,2’-oxydiethyl dicarbonate on reproduction and development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats.
The dose levels in this study were selected to be 0, 25, 60 and 115 mg/kg/day, based on the results of an oral 90-day repeated dose toxicity study and an oral Reproduction/Developmental Toxicity Screening Test in an attempt to produce graded responses to the test item. For the F0-generation, the following parameters and end points were evaluated: mortality/moribundity, clinical signs, body weight, food consumption, clinical pathology including measurement of thyroid hormones, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. For the F1-generation, the following parameters and end points were evaluated: mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, clinical pathology including measurement of thyroid hormones, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: estrous cycle determination, 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, macroscopy and measurement of thyroid hormones).
F0-generation - Parental results
A total of four animals were sacrificed prematurely. At 115 mg/kg/day, one male and two females were sacrificed in extremis on Days 77-95 based on their clinical condition and/or body weight loss. Correlating treatment related macroscopic and/or microscopic findings were noted in the liver, thymus and/or kidneys. At 60 mg/kg/day, one male was sacrificed in extremis on Day 66 based on its clinical condition. Macroscopically, grey-white foci were noted on the lungs, and misdosing was suspected. No microscopic findings were noted that could explain the moribund condition.
Microscopic findings of surviving animals were noted in the liver and spleen. Liver findings in males started at 60 mg/kg/day, and in females at 115 mg/kg/day. Findings were mainly concentrated in the periportal areas of the liver and represented a cascade of events related to hepatocellular toxicity. This cascade most likely started with test item-induced hepatocellular changes (basophilia and karyocytomegaly) and peribiliary infiltration of mononuclear cells followed by hepatocellular necrosis and a regeneration process expressed by presence of pigmented macrophages, fibrosis and bile duct hyperplasia. These findings correlated with increased liver enzyme activities and bile acids in males at 115 mg/kg/day. Particularly necrosis is degenerative in nature and therefore considered adverse, but also fibrosis may significantly affect the normal structure and function of the liver and is therefore also considered adverse.
In the spleen, an increased incidence and/or severity of extramedullary hematopoiesis in males starting at 60 mg/kg/day was observed, which could be related to changes in blood cell parameters (mainly increased white blood cell parameters, reticulocytes and platelets in males at 115 mg/kg/day). These changes were considered to be indirectly related to the liver findings due to a reaction on the necrosis and/or inflammation in the liver. Based on the relative low magnitude of the extramedullary hematopoiesis (compared to controls and background severities), these changes were considered non-adverse.
Clinical pathology changes a 115 mg/kg/day that were also noted, included decreased mean corpuscular volume and mean corpuscular haemoglobin and increase bilirubin concentrations (males) and increased red cell distribution width (both sexes). In absence of correlating microscopic findings these changes were considered non adverse.
Body weight (gain) in males at 115 mg/kg/day from Week 5 onwards was decreased. Based on the magnitude of the change this was considered non-adverse.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, food consumption, coagulation parameters and thyroid hormone levels).
F0-generation-Reproductive results
No reproduction toxicity was observed up to the highest dose level tested (115 mg/kg/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).
F0-generation-Developmental results
No developmental toxicity was observed up to the highest dose level tested (115 mg/kg/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 and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels and macroscopic examination).
F1-generation results
Microscopic findings were noted in the liver and spleen, which were comparable to the effects noted in the F0-generation.
Liver findings were noted in males and females at 115 mg/kg/day and in single females at 60 and 25 mg/kg/day. Findings were mainly concentrated in the periportal areas of the liver and represented a cascade of events related to hepatocellular toxicity. This cascade most likely started with test item-induced hepatocellular changes (basophilia and karyocytomegaly) and peribiliary infiltration of mononuclear cells followed by hepatocellular necrosis and a regeneration process expressed by presence of pigmented macrophages, fibrosis and bile duct hyperplasia. These findings correlated with increased clinical pathology liver parameters (liver enzymes and/or bile acids) observed at 115 mg/kg/day. Particularly necrosis is degenerative in nature and therefore considered adverse, but also fibrosis may significantly affect the normal structure and function of the liver and is therefore also considered adverse.
As the findings in the single females at 60 and 25 mg/kg/day were similar compared to the findings in multiple animals at 115 mg/kg/day, these findings were considered adverse in females starting at 25 mg/kg/day.
In the spleen, an increased incidence and/or severity of extramedullary hematopoiesis in males starting at 60 mg/kg/day and females at 115 mg/kg/day, which could be related to changes in blood cell parameters (mainly increased white blood cells, reticulocytes and platelets in males at 115 mg/kg/day). These changes were considered to be indirectly related to the liver findings due to a reaction on the necrosis and/or inflammation in the liver. Based on the relative low magnitude of the extramedullary hematopoiesis (compared to controls and background severities), these changes were considered non-adverse.
Clinical pathology changes at 115 mg/kg/day that were also noted included increased urea, inorganic phosphate and TSH concentrations (males and females), increased red blood cell concentrations and haematocrit (females), and decreased mean corpuscular haemoglobin (males). In the absence of correlating microscopic findings these changes were considered non-adverse.
The number of sperm cells was reduced in males at 115 mg/kg/day, while all other sperm parameters were unaffected. In females at 115 mg/kg/day estrus was affected, consisting of irregular (4/20 females), extended (1/20) and undetermined regularity (2/20). An irregular estrous cycle was also noted at 60 mg/kg/day (2/20). The effects on sperm cells and estrous cycle at 115 mg/kg/day were considered adverse based on the magnitude of the change or the incidence. These findings were noted at the high dose in which also systemic toxicity was noted including three F0-mortalities.
Sexual maturation at 115 mg/kg/day was slightly delayed for both sexes. The time to balanopreputial separation, and onset until vaginal opening and first estrus were increased. For males, body weights of the animals at balanopreputial separation were similar to control. Therefore, this delay was considered related to a slight delay in development and was considered not toxicologically relevant. For females, body weights at vaginal opening and first estrus were higher compared to control and a relation to treatment with the test item could not be excluded. However, as changes were minimal and as the periods remained within the historical control data, this was considered non-adverse.
The results of other endocrine disruptor measurements evaluated (thyroid hormone measurements, nipple retention, anogenital distance, and histopathology of endocrine organs) were considered not be affected by treatment with the test item.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, body weights, food consumption, coagulation parameters, splenic lymphocyte subpopulation and ovarian follicle counts).
In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohort 1), the following No Observed Adverse Effect Level (NOAELs) of Diallyl 2,2’-oxydiethyl dicarbonate were established:
NOAEL (general toxicity, F0) 25 mg/kg/day, based on test item related premature sacrifices at 115 mg/kg/day and adverse liver findings starting at 60 mg/kg/day.
NOAEL (general toxicity, F1) could not be established as adverse findings were noted in the liver starting at 25 mg/kg/day
NOAEL (reproduction, F0) >=115 mg/kg/day (no reproduction toxicity was observed up to the highest dose level tested)
NOAEL (reproduction, F1) 60 mg/kg/day (based on irregular estrous cycles and lower sperm counts; findings were noted at the high dose in which also systemic toxicity was noted including three F0-mortalities)
NOAEL (development) >= 115 mg/kg/day (no developmental toxicity was observed up to the highest dose level tested)
Effects on developmental toxicity
Description of key information
- Prenatal Developmental Toxicity Study (OECD 414, GLP), rats: NOAEL (maternal) 100 mg/kg/day; NOAEL (development) >= 100 mg/kg/day (highest evaluated dose), No effects on fetal observations.
- Teratology study (similar to OECD 414, GLP), rabbits: Embryotoxicity, accompanied by significant adverse effects upon maternal toxicity, was not considered to be of teratological significance.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 APR 2020 to 27 MAY 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- June 2018
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 1998
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 2008
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 24 hours at room temperature under normal laboratory light conditions and for at least 8 days in the refrigerator is confirmed over the concentration range 0.889 mg/g (1 mg/mL) to 177 mg/g (200 mg/mL) (solutions) - Species:
- rat
- Strain:
- Wistar
- Remarks:
- female Wistar Han Rats
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks old
- Weight at study initiation: 182 to 270 g
- Housing: Housed individually, in plastic cages containing appropriate bedding
- Diet (e.g. ad libitum): Pelleted rodent diet, provided ad libitum throughout the study, except during designated procedures
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20°C
- Humidity (%): 50 to 86 % (The values that were outside the targeted range occurred for 11 out of 22 days with a mean relative humidity of 72 to 86% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the
study.)
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
IN-LIFE DATES: From: 16/17 JUN 2020 (animal receipt) To: 09 JUL 2020 (Last date of necropsy) - Route of administration:
- oral: gavage
- Vehicle:
- polyethylene glycol
- Remarks:
- Polyethylene glycol 400
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
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 at least weekly, filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
VEHICLE
- Concentration in vehicle: 0, 10, 20, 40 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Dose Formulation Sample Collection Schedule:
- Occasion: Week 1 of treatment
- Concentration: All groups (The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.)
- Homogeneity: Groups 2 and 4 (The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results.)
All samples to be analyzed were transferred (at room temperature under normal laboratory light conditions) to the analytical laboratory at the Test Facility. Analyses were performed using a validated analytical procedure.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ±10% of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.
Formulation analyses confirmed that formulations of the test item in polyethylene glycol 400 were prepared accurately and homogenously. - Details on mating procedure:
- Time-mated females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating)
- Duration of treatment / exposure:
- Day 6 to Day 20 post-coitum, inclusive
- Frequency of treatment:
- Once daily oral gavage 7 days a week
- Duration of test:
- Day 6 post-coitum - scheduled necopsy: Day 21 post-coitum
- Dose / conc.:
- 0 mg/kg bw/day
- Remarks:
- Control
- Dose / conc.:
- 50 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 200 mg/kg bw/day
- No. of animals per sex per dose:
- 22 females/ group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The dose levels were selected based on the results of the dose range finder in the rat and the results of the 90-Day study in an attempt to produce graded responses to the test item.
- Other:
Justification of Route: The oral route of exposure was selected because this is the most appropriate route of administration for this substance for the assessment of reproductive toxicity and this was specified in the final decision by ECHA. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Animals were observed for general health/mortality and moribundity
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning on Day 2 post-coitum onwards up to the day prior to necropsy
BODY WEIGHT: Yes (weighed individually)
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum (The body weight of the animals of Subgroup 4 were performed on Day 3 post-coitum instead of Day 2 post-coitum. Body weight recorded for Subgroup 4 on Day 3 instead of Day 2 post-coitum were excluded from the data tables and were reported in the key to missing values. As dosing was only initiated on Day 6 post-coitum no data on possible test item effects are missing and based on the data of the other subgroups, sufficient information was available for evaluation).
Female No. 70 was also weighed on Day 11 post-coitum (in order to monitor the health status).
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.
Food consumption of the animals of subgroup 4 were performed on Day 3 post-coitum instead of Day 2 post-coitum. Food consumption data recorded for subgroup 4 on Day 3 instead of Day 2 post-coitum were excluded from the data tables and were reported in the key to missing values. As dosing was only initiated on Day 6 post-coitum no data on possible test item effects are missing and based on the data of the other subgroups, sufficient information was available for evaluation.
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post-coitum
Organ Weights: Liver, thyroid gland, and uterus were weighed at necropsy (except for females that delivered their offspring early, organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis). Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of one of the organs of a pair were taken and entered as a tissue comment. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated.
Necropsy: External, thoracic and abdominal examination
Histology: Thyroid glands of all animals were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Histopathology: Gland, thyroid, liver, stomach
Clinical Pathology: Thyroid Hormone Parameters (Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH)) - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gavid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Blood sampling:
- - Blood was collected on the day of scheduled necropsy (Blood for thyroid hormone analysis was only collected from animals surviving until scheduled necropsy)
- Animals were not fasted overnight
- Volume collected: 1.0 mL - Fetal examinations:
- - External examinations: Yes: Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities and their weight (not determined for fetuses of animals found dead, or sacrificed before planned necropsy)
- Soft tissue examinations (visceral): Yes: (one-half of the fetuses (live and dead) in each litter (all groups))
- Skeletal examinations: Yes: (one-half of the fetuses (i.e. the fetuses with heads)
- Head examinations: Yes (one-half of the fetuses in each litter)
- Anogenital distance (AGD): Yes: (all viable fetuses) - Statistics:
- Data Collected/Processed in ToxData:
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:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric:
- Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
- Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.
Incidence:
- An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
- No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss - Historical control data:
- Historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source are avialable.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Piloerection was observed for five females treated at 200 mg/kg/day on various days starting on Day 9 or 10 post-coitum (Female Nos. 70, 73 and 82) or on Day 21 post-coitum only (Female Nos. 69 and 87). In addition, Female No. 70 was scored having a pale appearance on Days 9-15 post-coitum and Female No. 87 was noted with a hunched posture on Day 21 post-coitum. As these clinical signs were only observed in females treated at 200 mg/kg/day and these findings are in line with the clinical signs of animals that died preterm, these were considered to be test item-related.
Salivation seen after dosing was observed in all females treated at 100 mg/kg/day and in the majority of the females treated at 200 mg/kg/day, starting after multiple days of dosing. This was considered of no toxicological relevance, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other findings that were noted included alopecia and scabs. These findings 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 with the test item.
No clinical signs were noted in the control group and at 50 mg/kg/day. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- In total, 12 females did not survive until scheduled necropsy.
The death of eight females was considered test item-related; all these females were treated at 200 mg/kg/day.
Five females treated at 200 mg/kg/day were euthanized for animal welfare reasons during the treatment period. For further details please refer to 'Details on results'. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- At 200 mg/kg/day, mean body weights were below the control mean during the whole study period, however only reaching statistical significance on Day 9 post-coitum (0.92x of control). Body weight gain was also lower on Day 9 post-coitum (mean body weight gain of 0% at 200 mg/kg/day vs. 4% in the concurrent control). For two females, body weight loss was noted between Day 18 and Day 21 post-coitum (Nos. 69 and 82). Overall, in females surviving until scheduled necropsy, body weight gain appeared to recover from Day 12 post-coitum onwards. At the end of the treatment period, body weights and corrected body weight gain were considered to be similar between the high dose group and concurrent control.
At 100 mg/kg/day, mean body weights were statistically significantly lower compared to control (up to 0.93x of control) on Days 6-12 post-coitum. However, mean body weight gain was comparable to the control group. This difference was most likely caused by the slightly lower mean body weight observed at 100 mg/kg/day compared to concurrent control on Day 2 post-coitum and was therefore considered unrelated to treatment with the test item.
At 50 mg/kg/day, no effects on body weight or body weight gain were observed - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- At 200 mg/kg/day, lower mean (relative) food consumption was observed compared to control females on Days 6-9 and 18-21 post-coitum (-20% and -24% of control, respectively). For the remaining period, mean food consumption was comparable to control.
At 50 and 100 mg/kg/day, lower mean absolute food consumption was observed on Days 2-6 post-coitum (prior to initiation of dosing, and therefore not test item-related; -9% and -13% of control, respectively) compared to concurrent control. Normal food consumption was observed from Day 6-9 post-coitum onwards. - Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At 200 mg/kg/day, mean serum levels of thyroid stimulating hormone (TSH) were statistically significantly higher compared to concurrent control (2.2x of control). Although mean TSH concentration remained within the available historical control data, the individual serum levels of 5/12^3 females were above the 95-percentile (P95) of the historical control data (all individual concurrent control data were within the historical control data).
At 100 mg/kg/day, slightly increased mean serum levels of TSH (1.3x of control) was also recorded, however without reaching statistical significance and mean values within historical control data. All individual data, except of Female No. 65 (0.887 mU/L), were within available historical control data. However, as this increase was observed in the mid- and high-dose groups and based on the magnitude of change, this might be related to treatment with the test item.
Mean serum levels of triiodothyronine (T3) were statistically significantly lower at 200 mg/kg/day (0.65x of control). Although mean T3 concentration remained within the available historical control data2, the individual serum levels of 4/12 females were below the 5-percentile (P5) of this historical control data (all individual concurrent control data were within the historical control data). Based on the magnitude of change, a relation to treatment with the test item could not be excluded.
Mean serum levels of total thyroxine (Total T4) were slightly lower (0.9x of control) in all treated groups when compared to concurrent control, however, without reaching statistical significance. In the absence of a dose-related response, this was considered to be unrelated to treatment with the test item. - Endocrine findings:
- not specified
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related significantly higher liver weights (absolute and/or relative to body weights) were noted in females as shown in the corresponding table under 'Any other information'. At 200 mg/kg/day, the absolute liver weight was increased by 23% and the relative liver weight by 30% compared to the control group.
There were no test item-related alterations in thyroid gland weights. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related macroscopic findings were present in the liver as:
- Focus/foci in 1/21 females treated at 50 mg/kg/day, in 1/22 females at 100 mg/kg/day and in 7/13 females at 200 mg/kg/day.
- Enlarged in 1/22 females treated at 100 mg/kg/day and in 6/13 females at 200 mg/kg/day.
- Irregular surface in 4/13 females treated at 200 mg/kg/day.
There were no test item-related gross thyroid gland observations. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- There were no test item-related microscopic thyroid gland observations.
All of the recorded microscopic findings in the thyroid gland 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. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Details on results:
- Mortality:
Female Nos. 74, 76, 79 and 81 were sacrificed in extremis on Day 9 post coitum based on body weight loss (between -9 and -12% compared to Day 6 post-coitum). Food consumption observed on Days 6-9 post-coitum was severely reduced in these females. For Female No. 76, clinical signs included hunched posture, piloerection and pale appearance on the day prior to necropsy. No clinical signs were observed for the other three females. At necropsy it was noted that the liver of all four females was pale, enlarged, hardened and/or had several dark red foci. In addition, the thymus of Female No. 76 was reduced in size, and Female No. 81 had isolated dark red foci on the outside of the stomach wall and ectopic splenic tissue.
Female No. 85 was sacrificed in extremis on Day 20 post-coitum based on clinical signs observed on this day. These clinical signs included lethargy, hunched posture, labored respiration and piloerection. Salivation was observed from Days 8-19 post-coitum. Normal body weight gain and food consumption was observed prior its sacrifice. Necropsy revealed a hardened liver with an irregular surface with many tan-colored foci.
Three females treated at 200 mg/kg/day were found dead on Day 20 or 21 post-coitum: Female Nos. 77 and 80 were found dead on the day of scheduled necropsy (Day 21 post-coitum). Both females were observed with normal body weight gain and food consumption up to Day 18 post-coitum. Subsequently, these females showed body weight loss (-8% and -5% compared to Day 18 post-coitum, respectively) and food consumption below normal values on Day 21 post-coitum. For Female No. 77, piloerection was observed on Days 10-14 and 21 post-coitum. Relevant clinical signs of Female No. 80 included hunched posture on Days 8-13 post-coitum. Both females showed autolysis at necropsy. Macroscopic findings included an irregular liver surface with many tan-colored foci (both females) and a hardened liver (Female No. 80 only).
Female No. 84 was found dead on Day 20 post-coitum. Normal body weight gain and food consumption was observed up to and including Day 15 post-coitum. However, a reduction in body weight of 11% compared to Day 15 post-coitum was observed on Day 18 post-coitum. In addition, food consumption on Day 15-18 post-coitum was below normal values. Clinical signs prior to its death included piloerection on Days 17-19 post-coitum and salivation from Day 8 post-coitum onwards. During necropsy, this female was observed being autolytic, which can be explained by the fact that this animal was already dead before necropsy. Other macroscopic findings included an irregular liver surface with many tan colored foci.
The females sacrificed for animal welfare reasons or that were found dead were all pregnant at the time of necropsy.
Three females were euthanized as they started to deliver their litters: Female No. 1 (control) and Female No. 23 (50 mg/kg/day) started to deliver on the day of scheduled necropsy (Day 21 post-coitum). Female No. 5 (control) was euthanized on Day 20 post-coitum, after it started to deliver its litter early. All females were observed with a normal body weight (gain) and food consumption prior to Day 21 post-coitum. In addition, no clinical signs or macroscopic findings were observed. Early deliveries are occasionally seen in this type of study. Given the absence of a dose-related trend and as it occurred in two control females, this was considered unrelated to treatment with the test item.
Female No. 5 (control) started to deliver its litter on Day 20 post-coitum and was therefore euthanized preterm. As a result, body weight (gain) and food consumption were not determined on Day 21 post-coitum, and corrected body weight gain and organs weights were not collected. Normal body weight (gain) and food consumption prior to Day 21 post-coitum were observed and no clinical signs or macroscopic findings were present.
In addition, Female No. 83 (200 mg/kg/day) was found dead on Day 9 post-coitum. Other than piloerection observed on Day 8 post-coitum, no clinical signs were observed prior to its death. At necropsy, this female had beginning autolysis. In addition, foamy contents in the trachea, the lungs were not collapsed and contained black foci and dark red foci on the liver were observed. Based on the lung findings, the death of this female was considered to be related to the gavage procedure. Therefore, this death was considered unrelated to treatment with the test item. - Number of abortions:
- no effects observed
- Description (incidence and severity):
- At scheduled necropsy, all females were pregnant, except for Female No. 2 (control group). Female No. 24 (50 mg/kg/day) was gravid but was observed with resorptions only. The females sacrificed for animal welfare reasons preterm or found dead (see information given under 'Mortality') were all pregnant at the time of necropsy.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- The number of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups up to 100 mg/kg/day were comparable and in the range of normal biological variation.
- Total litter losses by resorption:
- not specified
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- Early or late resorptions were considered not affected by treatment up to 100 mg/kg/day.
- Dead fetuses:
- effects observed, treatment-related
- Description (incidence and severity):
- Viable or dead fetuses were considered not affected by treatment up to 100 mg/kg/day.
- Changes in pregnancy duration:
- not specified
- Changes in number of pregnant:
- not specified
- Other effects:
- not examined
- Details on maternal toxic effects:
- Maternal toxicity: yes. Treatment with diallyl 2,2’-oxydiethyl dicarbonate resulted in a significant level of maternal toxicity at 200 mg/kg/day.
In the present study, the number of females with viable litters for evaluation was 19, 20, 22 and 12 in the control, 50, 100 and 200 mg/kg/day groups, respectively. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting (but kept in the raw data), to avoid bias and misinterpretation as the data at 200 mg/kg/day is incomplete. - Dose descriptor:
- NOAEL
- Remarks:
- general toxicity
- Effect level:
- 100 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- mortality
- Dose descriptor:
- NOAEL
- Remarks:
- maternal developmental toxicity
- Effect level:
- 100 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: Overall maternal developmental effects
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There were no test item-related effects on fetal body weights (both sexes) noted by treatment with the test item up to 100 mg/kg/day.
- Reduction in number of live offspring:
- not examined
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The male:female ratio was unaffected by treatment with the test item up to 100 mg/kg/day.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- There were no test item-related effects on litter size noted after treatment with the test item up to 100 mg/kg/day.
- Anogenital distance of all rodent fetuses:
- no effects observed
- Description (incidence and severity):
- There were no test item-related effects on fetal anogenital distance (both sexes) noted after treatment with the test item up to 100 mg/kg/day.
- Changes in postnatal survival:
- not examined
- External malformations:
- no effects observed
- Description (incidence and severity):
- No external malformations and external variations observed following treatment up to 100 mg/kg/day.
- Skeletal malformations:
- no effects observed
- Description (incidence and severity):
- There were no test item-related effects on skeletal morphology following treatment up to 100 mg/kg day.
In the control group, two fetuses were observed with skeletal malformations. Fetus No. A014-06 was observed with bent limb bones and Fetus No. A015-03 was observed with a vertebral centra anomaly. As both malformations only occurred in fetuses of the control group, these malformations were as such considered to be of spontaneous origin.
All skeletal variations occurred in the absence of a dose-related incidence trend, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data. Therefore, they were considered to be unrelated to treatment with the test item. - Visceral malformations:
- no effects observed
- Description (incidence and severity):
- There were no test item-related effects on visceral morphology following treatment up to 100 mg/kg/day.
At 50 mg/kg/day, two fetuses (Nos. A025-02 and A028-03) were observed with the malformation “small eye”, which was observed at soft tissue cephalic examination. Due to the occurrence at the low dose and as the % per litter remained within the maximum of the historical control data, the occurrence of this malformation was considered to be a chance finding.
The three visceral variations that were noted (small supernumerary liver lobes, dilated ureter and absent renal papilla) occurred at low incidences and in the control and/or 50 mg/kg/day groups only. Therefore, they were considered not related to treatment with the test item. - Other effects:
- not examined
- Details on embryotoxic / teratogenic effects:
- Embryoticity/ teratognic effects: No.
Up to 100 mg/kg/day, no test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations). - Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: overall embryotoxic / teratogenic effects
- Remarks on result:
- other: Absence of any test item-related effects on fetal observations
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Conclusions:
- Based on the results of this prenatal developmental toxicity study (i.e. adverse effects on body weight and food consumption and unscheduled deaths in dams treated at 200 mg/kg/day) the maternal No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being 100 mg/kg/day.
Due to the absence of any test item-related effects on fetal observations, the developmental No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being at least 100 mg/kg/day.
Note: In the present study, the number of females with viable litters for evaluation was 19, 20, 22 and 12 in the control, 50, 100 and 200 mg/kg/day groups, respectively, after exclusion of non-pregnant animals, animals showing an early delivery, and animals that did not survive until scheduled necropsy. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as this data is incomplete. - Executive summary:
The present study was conducted according to OECD 414 (adopted June 2018) and in compliance with GLP. In the study time-mated female Wistar Han rats were treated with diallyl 2,2’-oxydiethyl dicarbonate from Day 6 to 20 post-coitum, inclusive by daily oral gavage at dose levels of 50, 100 and 200 mg/kg/day. The rats of the control group received the vehicle, polyethylene glycol 400, alone.
The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (liver and thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.
Maternal toxicity
Treatment with diallyl 2,2’-oxydiethyl dicarbonate resulted in a significant level of maternal toxicity at 200 mg/kg/day. Test item-related mortality was observed at 200 mg/kg/day, consisting of five females that were euthanized for animal welfare reasons on Day 9 or 20 post-coitum and three females found dead on Day 20 or 21 post-coitum. All females euthanized in extremis were observed with body weight loss and food consumption below normal values and/or clinical signs including lethargy, hunched posture, labored respiration, piloerection and/or pale appearance.
Females that were found dead were observed with body weight loss and food consumption below normal values during the period adjacent to their death but with normal body weight gain and food consumption in the period before that. Clinical signs included piloerection or hunched posture. At necropsy, it was observed that all females had liver findings, including pale appearance of the liver, enlarged and/or hardened liver, dark red or tan colored foci on the liver and/or an irregular surface of the liver. In addition, isolated dark red foci on the outside of the stomach wall or a thymus that was reduced in size were observed in two of the females.
Piloerection was observed for five females treated at 200 mg/kg/day on various days starting on Day 9 or 10 post-coitum or was present on Day 21 post-coitum only. In addition, one female had a pale appearance on Days 9-15 post-coitum and another female showed a hunched posture on Day 21 post-coitum. As these clinical signs were only observed in females treated at 200 mg/kg/day and these findings are in line with the clinical signs of animals that died preterm, these effects were considered as an indication for an adverse test item-related effect.
Overall, mean food consumption was lower in females at 200 mg/kg/day on Days 6-9 and Days 18-21 post-coitum compared to control. For the remaining period, mean food consumption was comparable to control. In addition, mean body weight gain was statistically significantly lower on Day 9 post-coitum compared to control (which was most likely caused by the four females that were sacrificed for animal welfare reasons on Day 9 post-coitum).
For two females, body weight loss was noted between Day 18 and Day 21 post-coitum, indicating a similar pattern as the moribund animals. Despite the apparent recovery, given the size of the observed effects on body weight gain and food consumption resulting in sacrifice or death of eight high-dose females, these effects were considered adverse.
Macroscopic observation at necropsy revealed foci on the liver (statistically significant) as well as enlargement and/or hardening of the liver and an irregular surface of the liver for the majority of the remaining females at 200 mg/kg/day. These findings were also found in the females that were prematurely sacrificed due to body weight loss and food consumption below normal values and/or in the females that were found dead. In addition, liver weights (both absolute and relative to body weight) were also statistically significantly higher compared to concurrent control. Although histopathological evaluation was not performed, given the severity of these macroscopic findings they were considered adverse. Based on the low incidence of liver findings and the small magnitude of change in liver weights at 50 and 100 mg/kg/day, and in the absence of detrimental effects on animal welfare at these dose levels, the effects on the liver (presence of macroscopic findings and higher liver weights) were considered to be non-adverse. Higher mean serum levels of thyroid stimulating hormone (TSH) were observed at 100 and 200 mg/kg/day compared to concurrent control, reaching statistical significance at 200 mg/kg/day only. However, mean TSH concentrations remained within the available historical control data. Lower mean serum levels of triiodothyronine (T3) were observed in all treated groups compared to concurrent control, only reaching statistical significance at 200 mg/kg/day. However, mean T3 concentration remained within the available historical control data. No test item-related significant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. thyroxine (T4) and histopathologic examination of the thyroid gland). Excluding non-pregnant females, females that started to deliver their litter and females that did not survive until scheduled necropsy, there were 19 females in the control group, 20 females at 50 mg/kg/day, 22 females at 100 mg/kg/day and 12 females at 200 mg/kg/day with viable litters available for developmental evaluation. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting (but kept in the raw data), to avoid bias and misinterpretation as the data at 200 mg/kg/day is incomplete. The number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, and pre- and post-implantation loss were considered not affected by treatment up to 100 mg/kg/day.
Developmental toxicity
Up to 100 mg/kg/day, no test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).
In conclusion, based on the results of this prenatal developmental toxicity study (i.e. adverse effects on body weight and food consumption and unscheduled deaths in dams treated at 200 mg/kg/day) the maternal No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being 100 mg/kg/day. Note: In the present study, according to the OECD 414 guidelines, an insufficient number of litters was available for evaluation at 200 mg/kg/day. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as this data is incomplete.
Due to the absence of any test item-related effects on fetal observations, the developmental No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being at least 100 mg/kg/day.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1985-01-08 to 1985-02-08
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Remarks:
- This research was conducted at Bio-Research Laboratories Ltd. in compliance with the United States EPA Good Laboratory Practice Regulations (40 CFR Part 792). The study was performed prior to the adoption of the OECD Guideline 414 but all parameters described in the study are comparable to this Guideline method (Klimish et al. 1997).
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hazelton Dutchland Inc., Swampbridge Road, Denver, PA.
- Age at study initiation: 22-23 weeks
- Weight at study initiation: ranged from 3.2 to 4.3 kg
- Fasting period before study: no
- Housing: individually in mesh-bottomed stainless steel cages
- Diet (e.g. ad libitum): ad libitum (free access to pelleted chow (Purina Certified Rabbit Chow No. 5332, batches No. NOV 15842C, NOV 20841A, JAN 06842A, JAN 19841A, JAN 19842A).
- Water (e.g. ad libitum): ad libitum (free access to fresh municipal tap water)
- Acclimation period: 4 weeks prior to insemination.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 +- 3°C
- Humidity (%): 50 +- 20% (an occasional fluctuation of the relative humidity above 70% was not considered to have affected the outcome of the study)
- Air changes (per hr): data not available
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: To: data not available - Route of administration:
- dermal
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: not diluted. The doses administered was based on the most recent body weight of each rabbit.
PREPARATION FOR DOSAGE:
All animals were shaved on approximately day 0 of gestation. Hair was removed from an area of the dorsum at least 4 inches wide and 9 inches long.
METHOD OF DOSAGE
Groups of 18 inseminated rabbits were treated, by dermal application, with CR-39R monomer at dosages of 0.1, 0.5 or 1.0 mL/kg/day, for 6 hours per day, from day 6 to day 18 of gestation, inclusive. A control group was treated in an identical manner except that it was dosed with sterile isotonic saline at a volume of 1.0 mL/kg/day. The test liquid or control solution was spread on the shaved area of the back. Restraint collars were fitted on each animal immediately prior to dosage. After 6 hours, any residual compound or control solution was removed by blotting the dosage area with nonabsorbent cotton and the restraint collars were removed.
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at dose levels of 114, 572, and 1143 mg/kg/day
- Concentration (if solution): at respective dose volumes of 0.1, 0.5, and 1.0 mL/kg.
- Constant volume or concentration used: yes. The dose volumes were derived on the basis of the density of the test article, 1.143 grams/mL.
DIET PREPARATION Not applicable
CONTROL MATERIAL
The control article, sterile isotonic saline (0.9% sodiurn chloride for injection U.S.P.), supplied by Abbott Laboratories
- Amount: at the same dosage volume as treated animals in the high dose group (1 mL/kg bw/day)
- Lot/batch no. (if required): 54-542-DM-08 and 59-430-NA-3.
- Purity: no data - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Five-gram samples of CR-39R monomer were retained and subsequently shipped to the sponsor for analysis to confirm identity at the start of treatment, during the treatment period and at the end of the study. Viscosity measurements of the sample returned at the end of the study were within the normal specification.
- Details on mating procedure:
- - Impregnation procedure: [artificial insemination]
- If cohoused: not applicable
Female rabbits were luteinized with an intravenous injection of 50 IU of chorionic gonadotropin 19 days prior to insemination. Two to four hours prior to insemination, a second 50-1U dose of chorionic gonadotropin was administered to each female. Sperm samples were collected from stud bucks, and a diluted pooled sample was prepared. Each day samples from at least 4 males were used; in all a total of 18 males were utilized during the study. Each female was inseminated with at least 0.6 mL of the pooled sample, which contained in excess of 20 Mio spermatozoa/mL. Insemination was performed on January 2, 3, 7, 8, 9 and 10, 1985
- Verification of same strain and source of both sexes: data not available
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: data not available
- Any other deviations from standard protocol: no - Duration of treatment / exposure:
- from day 6 to day 18 of gestation, inclusive
- Frequency of treatment:
- daily for 6 hours
- Duration of test:
- 23 days, any surviving animals were terminated on day 29 of gestation
- Dose / conc.:
- 0.1 other: mL/kg bw/day
- Remarks:
- nominal conc. equivalent to 114 mg/kg bw/day
- Dose / conc.:
- 0.5 other: mL/kg bw/day
- Remarks:
- nominal conc. equivalent to 572 mg/kg bw/day
- Dose / conc.:
- 1 other: mL/kg bw/day
- Remarks:
- nominal conc. equivalent to 1143 mg/kg bw/day
- No. of animals per sex per dose:
- 18 female animals/ dose
- Control animals:
- other: 1mL/kg bw/day of 0.9% sodium chloride for injection U.S.P.
- Details on study design:
- - Dose selection rationale: Dosages were selected based upon the data from a pilot study entitled "A Range-Finding Teratology Study of CR-39® monomer by Dermal Application in the Rabbit" (Bio-Research Project No. 81981). In this study groups of 5 inseminated rabbits were treated by dermal application with CR-39R® monomer at dosages of 0.1, 0.5, 1.0 or 3,0 mL/kg/day 24 hours per day from day 6 to day 18 of gestation, inclusive. The dosage site was "occluded" with gauze and the animals were fitted with a restraining collar. Maternal toxicity was evidenced in all CR-39®-treated groups by skin reactions at the treatment site characterized by dark red/black areas which subsequently showed scab formation. Systemic toxicity was shown at the 3.0 mL/kg/day dose level where 1 rabbit was killed in poor condition and the remaining rabbits had significant weight loss following completion of dosage (day 18 to day 24 of gestation). Body weight losses were seen in all groups (treated and control) during the dosage period which suggest that these resulted from the dosage procedure (24-hour occlusion with restraining collars). Similarly, generally elevated abortion rates and increased incidences of late resorptions may have been related to this dosage procedure. Fetal malformations (hydrocephaly and abnormal limb/paw flexure) occurred among fetuses in the 0.1, 0.5 and 1.0 mL/kg/day groups (there were no live litters in the 3.0 mL/kg/day group).
- Rationale for animal assignment (if not random): randomized - Maternal examinations:
- All animals were checked for mortality or abnormal condition each day for any adverse reactions to treatment, indications of poor health and abnormal behavior from day 6 of gestation until day 18 of gestation. These examinations were performed prior to dosage and at least once post-dosing. In addition, the skin at the treatment site was examined daily. Any signs of abortion of premature delivery were recorded. Does which aborted or littered early were sacrificed. All aborted material was examined. All fetuses aborted prior to day 27 of gestation were examined externally and preserved in neutral buffered 10% formalin. All fetuses aborted on and after day 27 of gestation or littered early were examined as for fetuses at termination.
The overview of all examinations is outlined in Table 2 in "Any other information on materials and methods"
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Throughout the acclimation and gestation period
- Cage side observations checked in table [No.2] were included.
DETAILED CLINICAL OBSERVATIONS: Yes (Table 2)
- Time schedule: Throughout the acclimation and gestation period
BODY WEIGHT: Yes
- Time schedule for examinations: In addition to the weight assessment on arrival and weekly during the acclimation period, female rabbits were weighed on days 0, 6, 9, 12, 15, 18, 24 and 29 of gestation.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data Not applicable
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
Food consumption and fecal volume are qualitative assessed
-Calculation: not noted
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data. Not applicable
- Time schedule for examinations:
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: see Table 1 in "Any other information on materials and methods"
Any animal which was sacrificed during the study was killed and was then given a complete gross pathological
examination. The uterus was removed and examined. Any live or dead fetuses were examined externally and were preserved in neutral buffered formalin for does sacrificed prior to day 27 of gestation. For does examined on or after day 27 of gestation, live and dead fetuses were examined as for fetuses at cesarean section. Abnormal tissues were retained in neutral buffered 10% formalin.
OTHER:
Samples of tissue from the treatment site, two liver lobes and any abnormal tissues were preserved for further examinations - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
The uterus was removed, and the number and position of live fetuses, dead fetuses, empty implantation sites and early, middle and late resorptions were recorded. (Early resorption: metrial gland with some piacental/fetal tissue present. Middle resorption: both fetal and placental tissue present. Late resorption: recognizable fetal structure present. Empty implantation site: implantation site scar present, but no fetal or placental tissue.) - Fetal examinations:
- On day 29 of gestation the fetuses were removed from the uterus and weighed, and were then killed
- External examinations: Yes: [all per litter] and also any live or dead fetuses for does sacrificed prior to day 27 of gestation. For does examined on or after day 27 of gestation, live and dead fetuses were examined as for fetuses at cesarean section.
- Soft tissue examinations: Yes. Samples of tissue from two lobes of the liver from 1 male and 1 female fetus per litter were preserved in neutral buffered 10% formalin.
- Skeletal examinations: Yes [all per litter]
- Head examinations: Yes: [1/3 per litter ]
Other:
- Body weight: Yes [all per litter] - Statistics:
- Statistical analyses were performed upon both the body weights for all does and the body weights from only those pregnant with live litters at term. The group mean (S.D.) body weights and weight gains were calculated, and statistical analysis was performed using a one-way analysis of variance. Where the F value was found to be significant P <0.05), differences between control and treated groups were analyzed using Student's 't' test (continued in "Any other information on material and methods incl. tables").
- Indices:
- MATERNAL DATA: Pregnancy and abortion rates were calculated (see in "Any other information on material and methods incl. tables".)
UTERINE FINDINGS AND FETAL DATA: Pre- implantation loss (%) and post-implantation loss (%) (see in "Any other information on material and methods incl. tables".) - Historical control data:
- Historical control data (Rabbit NZW Dutchland 1983-1984 No. 81982) were provided in the key study study included in Appendix 11 and contain the same investigated endpoints as in the present study.
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Treatment of pregnant rabbits with CR-39® monomer at dosages of 0.1, 0.5 or 1.0 mL/kg/day by unoccluded dermal application resulted in decreased food intake, weight loss, abortion at 0.5 and 1.0 mL and mortality at the 1.0 mL/kg/day level. Necropsy findings among the 1.0 and 0.5 mL/kg bw/day treated group included a variety of adversed effects in organs as well as significant high abortion rates (continued in "Remarks on results") - Dose descriptor:
- LOEL
- Remarks:
- general toxicity
- Effect level:
- 114 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Remarks on result:
- other: equivalent to 0.1 mL/kg bw/day
- Dose descriptor:
- NOEL
- Remarks:
- maternal developmental toxicity
- Effect level:
- 114 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: overall maternal developmental effects
- Remarks on result:
- other: equivalent to 0.1 mL/kg bw/day
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes. Remark: only at doses with severe maternal toxicity
Details on embryotoxic / teratogenic effects:
Fetal findings in the 0.5 and 1.0 mL/kg/day groups of ocular opacities, small lens(es) and associated anomalies are indicative of embryotoxic effects during the later stages of ocular development. Also in the 0.5 mL/kg/day group incidental sceletal abnormalities were detected (continued in "Remarks on results"). - Dose descriptor:
- NOEL
- Effect level:
- 114 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: overall embryotoxicity
- Remarks on result:
- other: equivalent to 0.1 mL/kg bw/day
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Treatment of pregnant rabbits with CR-39® monomer at dosages of 0.1, 0.5 or 1.0 mL/kg/day by unoccluded dermal application resulted in decreased food intake, weight loss, abortion and mortality at the 1,0 mL/kg/day level.
Necropsy findings among the 1.0 mL/kg/day treated group animals included pale foci, firmness and/or irregular surface of the liver and, for the heart, kidneys and mesentery, pale foci and/or pale area(s). Maternal toxicity at the 0.5 mL/kg/day level was evidenced by lower food consumption and overall weight loss during the treatment periods (day 6 to day 18 of gestation). Also in the 0.5 mL/kg/day group 3 rabbits aborted, and gross pathological findings in the liver (firmness/irregular surface) and mesentery (pale foci) each occurred once. Some rabbits in all CR-39 monomer-treated groups had skin lesions, reddening, dark red/black areas and scab formation. Uterine findings for pregnant does surviving to day 29 of gestation, including 3 rabbits in the 1.0 mL/kg/day, were unaffected.
However, among rabbits in the 1.0 mL/kg/day group which aborted, died or were sacrificed in a moribund condition, there was a high incidence of resorptions indicative of embryolethality. Fetal findings in the 0.5 and 1.0 mL/kg/day groups of ocular opacities, small lens(es) and associated anomalies are indicative of embryotoxic effects during the later stages of ocular development. Also in the 0.5 mL/kg/day group the incidence of single thirteenth ribs was decreased and the occurrence of paired thirteenth ribs was slightly increased, combined with an increased incidence of 27 presacral vertebrae. These skeletal findings, when accompanying adverse effects upon maternal weight gain, are not considered to be of teratological significance. Among abortuses in the 1.0 mL/kg/day group, eventration of the intestines and liver were common observations. While these may be related to treatment, there is the possibility that they were related to trauma associated with the abortion process. There was no evidence of embryolethality or embryotoxicity at the 0.1 mL/kg/day dose level. - Executive summary:
CR-39 ® monomer, a (Methylene glycol carbonate ester), was administered by dermal application to groups of gravid rabbits throughout major organogenesis to assess its teratogenic potential.
Groups of 18 inseminated rabbits were treated, by dermal application, with CR-39 ® monomer at dosages of 0.1, 0.5 or 1.0 mL/kg/day (equivalent to 114, 572 and 1143 mg/kg bw/day, respectively), for 6 hours per day, from day 6 to day 18 of gestation, inclusive. A control group was treated in an identical manner except that it was dosed with sterile isotonic saline at a volume of 1.0 mL/kg/day. Treatment related adverse effects are summarized in Table 1 in "Overall remarks, attachments". Six rabbits died and 1 was killed in a moribund condition in the 1.0 mL/kg/day treated group. No other animals died or were sacrificed in a moribund condition. There were skin lesions at the dosage sites for some rabbits in all CR-39 ® monomer-treated groups which were typified by dark red/black thickened areas which subsequently turned yellow and showed scab formation. The area affected was related to dosage. The incidence of rabbits not eating and/or having decreased fecal volume (quantitatively assessed) was increased in the 0.5 and 1.0 mL/kg/day group. The 1.0 mL/kg/day group had significant weight losses during all intervals in the treatment period (day 6 to day 18 of gestation) and between days 18 and 24 of gestation. In consequence the body weights were significantly less than those of the control group on days 12, 15, 18 and 24 of gestation. There was a marked weight loss between days 6 and 18 of gestation in the 0.5 mL/kg/day group. The growth of the 0.1 mL/kg/day group was similar to that of the controls. Pale foci, firmness and/or an irregular surface were common findings for the liver of animals in the 1.0 mL/kg/day group. Other findings seen at necropsy for this group (1.0 mL/kg/day) included pale foci or pale area(s) on the heart, kidneys and/or mesentery of some rabbits. In the 0.5 mL/kg/day group 1 rabbit had firmness and an irregular surface to the liver and a second rabbit had pale foci on the mesentery. One, 0, 3 and 6 does in the control, 0.1, 0.5 and 1.0 mL/kg/day treated groups, respectively, aborted. The pregnancy rate in all groups was at least 88.9%. The ovarian and uterine parameters (number of corpora lutea, implantation sites, live fetuses, dead fetuses, resorptions, fetal weights and pre- and post- implantation losses) of the 0.1 and 0.5 mL/kg/day treated does were unaffected. For the 1.0 mL/kg/day group there was a high level of resorptions observed for animal s dying, aborting, Uttering early or sacrificed preterminally, but the uterine parameters of the 3 females in this group alive on day 29 of gestation were similar to control values.
In the 0.5 and 1.0 mL/kg/day groups significant (P <0.01 and P <0.001, respectively) numbers of fetuses had small lens(es), In the 0.5 mL/kg/day group 6 -fetuses from 3 litters were affected and in the 1.0 mL/kg/day group 5 fetuses from 1 litter were affected. All the affected fetuses in the 1.0 mL/kg/day group and some in the 0.5 mL/kg/day group also had ocular opacities. Also among these fetuses there were other ocular findings. The overall incidence of minor visceral anomalies was not significantly different from control values. The incidences of major malformations and minor skeletal anomalies among term fetuses were not significantly different from control values. Findings of eventration of the liver and intestines among abortuses from 3 litters in the 1.0 mL/kg/day group were possibly related to trauma in the abortion process. The incidence of major malformations in the treated groups, among 1itters from females examined at cesarean section, was not significantly different from control values. In the 0.5 mL/kg/day group there was a significant (P <0.05) decrease in the incidence of single thirteenth ribs and an accompanying increase in the incidence of paired thirteenth ribs and a significant increase in the incidence of 27 presacral vertebrae. The incidence of sternebral variants in the treated groups was not significantly different from control values.
CR-39 ® monomer was found to produce maternal toxicity in terms of skin lesions at the treatment site at levels of 0.1, 0.5 and 1.0 mL/kg/day. Systemic toxicity occurred at the 0.5 and 1.0 mL/kg/day group, with the majority of rabbits in the 1.0 mL/kg/day group either dying or aborting. Maternal toxicity at the 0.5 and 1.0 mL/kg bw evidenced by body weight loss. Embryotoxicity was evidenced by significantly increased ocular anomalies in the 0.5 and 1.0 mL/kg/day groups. These findings when accompanying significant adverse effects upon maternal weight loss, are not considered to be of teratological significance. There was some indication of embyrolethality among those rabbits in the 1.0 mL/kg/day group which died or aborted. Neither embryolethality nor embroyotoxicity occurred at the 0.1 mL/kg/day dose level.
Referenceopen allclose all
Results
Dose Formulation Analyses:
- Accuracy: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations.
- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Organ Weights:
| Dose level (mg/kg/day): | 50 | 100 | 200 |
| LIVER | |||
| Absolute | 4 | 5 | 23** |
| Relative to body weight | 6 | 9* | 30** |
*: P<0.05, **: P<0.01
Fetal Findings: In the present study, the number of females with viable litters for evaluation was 19, 20, 22 and 12 in the control, 50, 100 and 200 mg/kg/day groups, respectively. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting (but kept in the raw data), to avoid bias and misinterpretation as the data at 200 mg/kg/day is incomplete.
Mortality
In the 1.0 mL/kg/day treated group, 6 rabbits died and 1 was killed in a moribund condition.
Three of these rabbits (Nos. 457, 461 and 468) died after 1 or 2 days of treatment. Clinical observations for rabbit 461 included not eating and a red wet area of scab formation at the dosage site; there were no clinical findings for rabbits 457 and 468. Gross pathological findings common to all 3 rabbits were dark area(s) on the mucosa of the stomach, red discoloration or clot in the vaginal serosa and dilatation or red discoloration of the urinary bladder. For rabbit 468 pale areas were noted on the endocardium and pale foci on the cortices of the kidneys. Rabbits 453, 463 and 464 died on days 22, 21 and 20 of gestation, respectively. All three rabbits had not been eating at times during the dosage period, and on the 2 days prior to death rabbit 463 had been weak and thin and on the day prior to death rabbit 464 had labored respiration. At necropsy, findings in the stomachs included pale/dark/ depressed area(s) on the mucosa of the stomach for all those rabbits; there was a perforation in the stomach of rabbit 453. The digesta of rabbit 463 was dark and firm. Rabbit 464 had dark frothy fluid in the trachea and uncollapsed lungs which were reddened; in the thoracic cavity of rabbit 453 there was brownish fluid. Both rabbits 463 and 464 had generalized pallor and firmness of the liver with multiple pale foci; in addition, rabbit 463 had a yellowish pale area extending into the parenchyma. Rabbit 463 had multiple dark areas on the kidneys. Rabbit 460 was killed in a moribund condition of day 22 of gestation. Clinical signs on this day included yellow nasal discharge, labored respiration, coldness and prostration. Necropsy observations included dry thickened areas at the dose site, pale foci on the left lobe of the liver, multiple dark depressed areas on the mucosae of the stomach and duodenum and dark firm digesta in the large intestine.
Clinical Findings
Among rabbits in the treated groups, dark red/black discrete or diffuse areas, sometimes preceded by reddening of the skin, were commonly seen at the dosage sites. For some rabbits in all CR39 ® monomer-treated groups, red secretions from the black thickened areas were noted. These lesions often subsequently developed yellow coloration at their periphery and scab formation. The area of the dosage site affected by black thickened lesions was increased in a dose-related fashion (it should be noted that dosage was varied by adjustment of dose volume). Qualitative assessment of food consumption and fecal volume indicated dose-related increased incidences in the 0.5 and 1.0 mL/kg/day dose groups during the treatment period and for the period day 19 to day 24 of gestation. Reddening of the dorsal cervical region and pinna was seen for some rabbits in the 1.0 mL/kg/day group. One doe in the 0.5 mL/kg/day group and 2 does in the 1.0 mL/kg/day group had reddening or swelling in the urogenital area.
Body weights
Rabbits (including all animals in the group) in the 1.0 mL/kg/day group had significant weight losses throughout the dosage period (day 6 to day 18 of gestation), with weight losses continuing between days 18 and 24 of gestation. These weight losses resulted in significantly lower body weights on days 12, 15, 18 and 24 of gestation. Consideration of all rabbits in the 0.5 mL/kg/day group showed weight losses or slightly lower body weight gains than the control group during assessment intervals of the dosage period, resulting in a significant (P<0.01) overall weight loss between days 6 and 18 of gestation. For those rabbits in the 0.5 mL/kg/day group which had live litters on day 29 of gestation, there was an overall weight loss between days 6 and 18 of gestation. The body weights and body weight gains of the 0.1 mL/kg/day treated group were similar to control values.
Gross pathological findings
Two, 0, 3 and 8 rabbits were sacrificed preterminally due to abortion or littering early in the control, 0.1, 0.5 or 1.0 mL/kg/day groups, respectively. A further 6 rabbits died and 1 rabbit was sacrificed in a moribund condition in the 1.0 mL/kg/day group. The remaining rabbits were examined at cesarean section on day 29 of gestation. Pale foci on the liver were noted for 5 rabbits in the 1.0 mL/kg/day treated group dying or sacrificed between days 20 and 22 of gestation. Firmness and/or an irregular surface of the liver occurred for 1 doe in the 0.5 mL/kg/day group and 4 does in the 1.0 mL/kg/day group which died or were sacrificed. Four rabbits in the 1.0 mL/kg/day group dying or sacrificed preterminally had pale foci or pale area(s) on the heart. Three of these 4 rabbits and 1 further rabbit in this group had pale foci or pale areas on the kidneys. Pale foci or pale areas on the mesentery occurred for 1 doe in the 0.5 mL/kg/day group and 4 does in the 1.0 mL/kg/day group. In all CR39R ® monomer-treated groups skin lesions including areas of the dosage site which were dark, pale, depressed, dry, thickened and/or with scab formation were noted at necropsy. Some animals in all groups had findings in the stomach which included dark areas/foci, depressed areas (ulceration) and/or thickening. There was a slightly higher incidence of these findings in the 1.0 mL/kg/day group which may be correlated with the greater occurrence of rabbits not eating in this group. (It is also worthy of note that the majority of animals in the 1.0 mL/kg/day dose group with stomach lesions had been found dead.) Dark ingesta/digesta, probably indicative of bleeding from areas of ulceration, was noted for 4 rabbits in the 1.0 mL/kg/day group, and 1 other rabbit had a perforation of the stomach. The incidence of prominent lobular architecture and pallor in the control and treated groups indicated these findings were incidental. The remaining gross pathological findings were judged to be incidental or agonal.
Abortion
One, 0, 3 and 6 does in the control, 0.1, 0.5 and 1.0 mL/kg/day dose group, respectively, aborted. In addition, 1 doe in the control group and 2 does in the 1.0 mL/kg/day treated group littered early.
Details on maternal toxic effects (Uterine findings)
The pregnancy rate was at least 88.9% in all groups. There were 3 females in the 1.0 mL/kg/day dose group examined on day 29 of gestation, the remainder having died or been sacrificed due to abortion, littering early or poor condition. The ovarian and uterine parameters including numbers of corpora lutea, implantation sites, live fetuses, dead fetuses and resorptions, the fetal and litter weights, uterine weights and the pre- and post-impl antation losses in the 0.1 and 0.5 mL/kg/day treated groups were similar to control values for those does alive on day 29 of gestation. Two of the 3 does which aborted in the 0.5 mL/kg/day group had a number of resorptions in the uterus. For those does which aborted, littered early, died or were sacrificed preterminally in the 1.0 mL/kg/day treated groups, resorptions were frequently observed either as aborted material or in utero.
Fetal findings
a) Major Malformations
The incidence of major malformations in the treated groups, among 1itters from females examined at cesarean section, was not significantly different from control values.
b) Minor Visceral Anomalies
A significantly lower overall incidence of fetuses with minor visceral anomalies in the 0.1 mL/kg/day dosed group was considered to be unrelated to dosage. There was a significant (P<0.001) increase in the incidence of ocular opacities and small lenses in the 1.0 mL/kg/day treated group 5 fetuses from 1 of the 3 litters examined at term being affected. Three of these fetuses also had lens(es) formed in 2 layers. A significant (P<0.01) number of fetuses (6 fetuses from 3 litters) in the 0.5 ml/kg/day group had small lens(es). Some of these fetuses had opacities of the lens (an external observation), lens(es) formed in 2 layers, encapsulated with or connected to retinoid/choroid/corneal tissue. c) Minor Skeletal Anomalies
The overall incidences of litters and fetuses affected in the treated groups were unaffected. Significantly (P<0.05) elevated incidences of fetuses with absent pubic bones and reduced numbers of phalanges and/or metatarsals in the hindpaws in the 0.1 mL/kg/day treated group were not attributed to dosage as all the affected fetuses were from 1 litter.
d) Common Skeletal Variants
The incidence of sternebral variants in the treated groups was not significantly different from control values. There was a significant (P< 0.05) decrease in the occurrence of single thirteenth ribs and a concomitant increase in the incidence of paired thirteenth ribs in the 0.5 mL/kg/day along with a significant (P<0.05) elevation in the incidence of 27 presacral vertebrae.
e) Fetuses from Aborted Litters
Among abortuses recovered on days 19 to 22 of gestation from the 1.0 mL/kg/day group, open eye(s), domed skull and eventration of the intestines and liver at the umbilicus were common findings. Two abortuses (454/1 and 3) had clefts in the vertebral column. Since the eyelids are normally closed by day 20 of gestation, this finding in fetuses aborted and found dead, which may have been dead in utero, may be indicative only of their stage of development. Similarly the findings of domed skull (fetuses were examined on criteria for fetuses on day 29 of gestation) could be reflective of their gestational age. In considering the remaining observations, the possibility that these findings may have resulted from traumatic injury precludes a definitive assessment of their relationship to CR39 ®-monomer treatment.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 100 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 114 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
Additional information
A Prenatal Developmental Toxicity Study was conducted according to OECD 414 (adopted June 2018) and in compliance with GLP. In the study time-mated female Wistar Han rats were treated with diallyl 2,2’-oxydiethyl dicarbonate from Day 6 to 20 post-coitum, inclusive by daily oral gavage at dose levels of 50, 100 and 200 mg/kg/day. The rats of the control group received the vehicle, polyethylene glycol 400, alone.
The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), gross necropsy findings, organ weights (liver and thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.
Maternal toxicity
Treatment with diallyl 2,2’-oxydiethyl dicarbonate resulted in a significant level of maternal toxicity at 200 mg/kg/day. Test item-related mortality was observed at 200 mg/kg/day, consisting of five females that were euthanized for animal welfare reasons on Day 9 or 20 post-coitum and three females found dead on Day 20 or 21 post-coitum. All females euthanized in extremis were observed with body weight loss and food consumption below normal values and/or clinical signs including lethargy, hunched posture, labored respiration, piloerection and/or pale appearance.
Females that were found dead were observed with body weight loss and food consumption below normal values during the period adjacent to their death but with normal body weight gain and food consumption in the period before that. Clinical signs included piloerection or hunched posture. At necropsy, it was observed that all females had liver findings, including pale appearance of the liver, enlarged and/or hardened liver, dark red or tan colored foci on the liver and/or an irregular surface of the liver. In addition, isolated dark red foci on the outside of the stomach wall or a thymus that was reduced in size were observed in two of the females.
Piloerection was observed for five females treated at 200 mg/kg/day on various days starting on Day 9 or 10 post-coitum or was present on Day 21 post-coitum only. In addition, one female had a pale appearance on Days 9-15 post-coitum and another female showed a hunched posture on Day 21 post-coitum. As these clinical signs were only observed in females treated at 200 mg/kg/day and these findings are in line with the clinical signs of animals that died preterm, these effects were considered as an indication for an adverse test item-related effect.
Overall, mean food consumption was lower in females at 200 mg/kg/day on Days 6-9 and Days 18-21 post-coitum compared to control. For the remaining period, mean food consumption was comparable to control. In addition, mean body weight gain was statistically significantly lower on Day 9 post-coitum compared to control (which was most likely caused by the four females that were sacrificed for animal welfare reasons on Day 9 post-coitum).
For two females, body weight loss was noted between Day 18 and Day 21 post-coitum, indicating a similar pattern as the moribund animals. Despite the apparent recovery, given the size of the observed effects on body weight gain and food consumption resulting in sacrifice or death of eight high-dose females, these effects were considered adverse.
Macroscopic observation at necropsy revealed foci on the liver (statistically significant) as well as enlargement and/or hardening of the liver and an irregular surface of the liver for the majority of the remaining females at 200 mg/kg/day. These findings were also found in the females that were prematurely sacrificed due to body weight loss and food consumption below normal values and/or in the females that were found dead. In addition, liver weights (both absolute and relative to body weight) were also statistically significantly higher compared to concurrent control. Although histopathological evaluation was not performed, given the severity of these macroscopic findings they were considered adverse. Based on the low incidence of liver findings and the small magnitude of change in liver weights at 50 and 100 mg/kg/day, and in the absence of detrimental effects on animal welfare at these dose levels, the effects on the liver (presence of macroscopic findings and higher liver weights) were considered to be non-adverse. Higher mean serum levels of thyroid stimulating hormone (TSH) were observed at 100 and 200 mg/kg/day compared to concurrent control, reaching statistical significance at 200 mg/kg/day only. However, mean TSH concentrations remained within the available historical control data. Lower mean serum levels of triiodothyronine (T3) were observed in all treated groups compared to concurrent control, only reaching statistical significance at 200 mg/kg/day. However, mean T3 concentration remained within the available historical control data. No test item-related significant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. thyroxine (T4) and histopathologic examination of the thyroid gland). Excluding non-pregnant females, females that started to deliver their litter and females that did not survive until scheduled necropsy, there were 19 females in the control group, 20 females at 50 mg/kg/day, 22 females at 100 mg/kg/day and 12 females at 200 mg/kg/day with viable litters available for developmental evaluation. As according to the guidelines, a minimum of 16 litters is required for evaluation of developmental data, the 12 litters available at 200 mg/kg/day were not sufficient for a robust and valid evaluation of developmental data. As such, developmental data of Group 4 were excluded from reporting (but kept in the raw data), to avoid bias and misinterpretation as the data at 200 mg/kg/day is incomplete. The number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, and pre- and post-implantation loss were considered not affected by treatment up to 100 mg/kg/day.
Developmental toxicity
Up to 100 mg/kg/day, no test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).
In conclusion, based on the results of this prenatal developmental toxicity study (i.e. adverse effects on body weight and food consumption and unscheduled deaths in dams treated at 200 mg/kg/day) the maternal No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being 100 mg/kg/day. Note: In the present study, according to the OECD 414 guidelines, an insufficient number of litters was available for evaluation at 200 mg/kg/day. As such, developmental data of Group 4 were excluded from reporting, to avoid bias and misinterpretation as this data is incomplete.
Due to the absence of any test item-related effects on fetal observations, the developmental No Observed Adverse Effect Level (NOAEL) for diallyl 2,2’-oxydiethyl dicarbonate was established as being at least 100 mg/kg/day.
In another teratology study (OECD 414), CR-39®monomer, a (Methylene glycol carbonate ester), was administered by dermal application to groups of gravid rabbits throughout major organogenesis to assess its teratogenic potential (Bio-Research Laboratories, 1985).
Groups of 18 inseminated rabbits were treated, by dermal application, with CR-39®monomer at dosages of 0.1, 0.5 or 1.0 mL/kg/day (114, 572 and 1143 mg/kg bw/day), for 6 hours per day, from day 6 to day 18 of gestation, inclusive. A control group was treated in an identical manner except that it was dosed with sterile isotonic saline at a volume of 1.0 mL/kg/day. Six rabbits died and 1 was killed in a moribund condition in the 1.0 mL/kg/day treated group. No other animals died or were sacrificed in a moribund condition. There were skin lesions at the dosage sites for some rabbits in all CR-39®monomer-treated groups which were typified by dark red/black thickened areas which subsequently turned yellow and showed scab formation. The area affected was related to dosage. The incidence of rabbits not eating and/or having decreased fecal volume (quantitatively assessed) was increased in the 0.5 and 1.0 mL/kg/day group. The 1.0 mL/kg/day group had significant weight losses during all intervals in the treatment period (day 6 to day 18 of gestation) and between days 18 and 24 of gestation. In consequence the body weights were significantly less than those of the control group on days 12, 15, 18 and 24 of gestation. There was a marked weight loss between days 6 and 18 of gestation in the 0.5 mL/kg/day group. The growth of the 0.1 mL/kg/day group was similar to that of the controls. Pale foci, firmness and/or an irregular surface were common findings for the liver of animals in the 1.0 mL/kg/day group. Other findings seen at necropsy for this group (1.0 mL/kg/day) included pale foci or pale area(s) on the heart, kidneys and/or mesentery of some rabbits. In the 0.5 mL/kg/day group 1 rabbit had firmness and an irregular surface to the liver and a second rabbit had pale foci on the mesentery. One, 0, 3 and 6 does in the control, 0.1, 0.5 and 1.0 mL/kg/day treated groups, respectively, aborted. The pregnancy rate in all groups was at least 88.9%. The ovarian and uterine parameters (number of corpora lutea, implantation sites, live fetuses, dead fetuses, resorptions, fetal weights and pre- and post- implantation losses) of the 0.1 and 0.5 mL/kg/day treated does were unaffected. For the 1.0 mL/kg/day group there was a high level of resorptions observed for animal s dying, aborting, Uttering early or sacrificed preterminally, but the uterine parameters of the 3 females in this group alive on day 29 of gestation were similar to control values.
In the 0.5 and 1.0 mL/kg/day groups significant (P <0.01 and P <0.001, respectively) numbers of fetuses had small lens(es), In the 0.5 mL/kg/day group 6 -fetuses from 3 litters were affected and in the 1.0 mL/kg/day group 5 fetuses from 1 litter were affected. All the affected fetuses in the 1.0 mL/kg/day group and some in the 0.5 mL/kg/day group also had ocular opacities. Also among these fetuses there were other ocular findings. The overall incidence of minor visceral anomalies was not significantly different from control values. The incidences of major malformations and minor skeletal anomalies among term fetuses were not significantly different from control values. Findings of eventration of the liver and intestines among abortuses from 3 litters in the 1.0 mL/kg/day group were possibly related to trauma in the abortion process. The incidence of major malformations in the treated groups, among 1itters from females examined at cesarean section, was not significantly different from control values. In the 0.5 mL/kg/day group there was a significant (P <0.05) decrease in the incidence of single thirteenth ribs and an accompanying increase in the incidence of paired thirteenth ribs and a significant increase in the incidence of 27 presacral vertebrae. The incidence of sternebral variants in the treated groups was not significantly different from control values.
CR-39®monomer was found to produce maternal toxicity in terms of skin lesions at the treatment site at levels of 0.1, 0.5 and 1.0 mL/kg/day. Systemic toxicity occurred at the 0.5 and 1.0 mL/kg/day group, with the majority of rabbits in the 1.0 mL/kg/day group either dying or aborting. Maternal toxicity at the 0.5 and 1.0 mL/kg bw evidenced by body weight loss. Embryotoxicity was evidenced by significantly increased ocular anomalies in the 0.5 and 1.0 mL/kg/day groups. These findings when accompanying significant adverse effects upon maternal weight loss, are not considered to be of teratological significance. There was some indication of embyrolethality among those rabbits in the 1.0 mL/kg/day group which died or aborted. Neither embryolethality nor embroyotoxicity occurred at the 0.1 mL/kg/day dose level.
Justification for classification or non-classification
In a highly reliable EOGRTS according to the OECD TG 443 and GLP no developmental or reproductive toxicity was observed in the parental (F0) generation up to the highest dose level tested (115 mg/kg/day). In the F1 generation irregular estrous cycles and lower sperm counts were recorded at 115 mg/kg/day, however, these effects occurred at the high dose in which also systemic toxicity was noted. In the F0 generation the dose level of 115 mg/kg/day caused systemic toxicity, including severe liver findings and three animals were even sacrificed in extremis after 77 to 95 days of treatment. Hence, it cannot be excluded that the observed findings in number of sperm cells and estrous cycle at the high dose group of F1 animals are secondary to the observed systemic toxicity.
Liver toxicity was also noted in the F1 generation already noted at the lowest dose level of 25 mg/kg/day. These liver findings have confirmed a suspected specific target organ toxicity and triggered a corresponding classification (please refer to the endpoint summary of the repeated dose toxicity section).
Neither adverse effects on gonads nor changes in reproductive performance and fertility were found in the Combined Repeat Dose Toxicity Study with a Reproductive /Developmental Screening Test according to OECD 422 (MPI Research, 2005) and signs of embryolethality and embryotoxicity as well as adverse effects on fertility of rabbits in the teratology study (Bio-Research Laboratories, 1985) are the consequences of severe systemic toxicity.
These findings are supported by the Prenatal Developmental Toxicity Study conducted according to OECD TG 414 and in compliance with GLP. This study demonstrated severe signs of systemic toxicity and the absence of any test item-related effects on fetal observations in Han Wistar rats.
Taken together, a classification according to the criteria of Regulation (EC) No 1272/2008 is not warranted.
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