Reaction mass of 3-({[2,2-dimethyl-3-({[2-(2-{[(prop-2-en-1-yloxy)carbonyl]oxy}ethoxy)ethoxy]carbonyl}oxy)propoxy]carbonyl}oxy)prop-1-ene and 3-{[(2,2-dimethyl-3-{[(prop-2-en-1-yloxy)carbonyl]oxy}propoxy)carbonyl]oxy}prop-1-ene and diallyl 2,2'-oxydiethyl dicarbonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

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:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

No

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diallyl 2,2'-oxydiethyl dicarbonate

EC Number:

205-528-7

EC Name:

Diallyl 2,2'-oxydiethyl dicarbonate

Cas Number:

142-22-3

Molecular formula:

C12H18O7

IUPAC Name:

diallyl 2,2'-oxydiethyl dicarbonate

Details on test material:

- Name of test material (as cited in study report): diallyl diglycol carbonate (ADC)
The test article was used as received from the Sponsor and no adjustment was made for purity.
- Substance type: pure active substance
- Physical state: liquid
- Purity test date: 2002-11-19
- Stability under test conditions: considered stable for study duration
- Storage condition of test material: at room temperature

Test animals

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.

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
150 mg/kg bw/day
Basis:
other: dermal

Doses / concentrations 2

Remarks:

Doses / Concentrations:
454 mg/kg bw/day
Basis:
other: dermal

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1030 mg/kg bw/day
Basis:
other: dermal

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 030 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

VIABILITY (OFFSPRING)
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.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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 7.5.2 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.