A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 30, 2010 to January 31, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

EC Number:

412-260-6

EC Name:

A mixture of: 7,9,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecane-1,16-diylprop-2-enoate; 7,7,9-trimethyl-3,14-dioxa-4,13-dioxo-5,12-diazahexadecan-1,16-diylprop-2-enoate

Cas Number:

52658-19-2

Molecular formula:

C21H34N2O8 (molecular formula of the two isomers constituting the substance)

IUPAC Name:

2-({[(2R)-2,4,4-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[(2S)-2,4,4-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[(3R)-3,5,5-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[(3S)-3,5,5-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[2,2,4-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate; 2-({[2,4,4-trimethyl-6-({[2-(prop-2-enoyloxy)ethoxy]carbonyl}amino)hexyl]carbamoyl}oxy)ethyl prop-2-enoate

Test material form:

liquid: viscous

Remarks:

yellowish liquid

Details on test material:

- Name of test material (as cited in study report): Ebecryl® 4858
- Lot/batch No.: JBHF0121T
- Expiration date of the lot/batch: 7 April 2012
- Stability under test conditions: stable
- Storage condition of test material: At room temperature protected from light

Test animals

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: (P) 12 wks; (F1) x wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:

- Housing: Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material was supplied.
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).

- Diet: Free access to pelleted rodent diet
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 – 23.0
- Humidity (%): 40-72
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

specific gravity 1.125

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared on a weekly basis and were homogenised to a visually acceptable level on a daily basis. Adjustment was made for specific gravity of the vehicle (1.125) and test substance (1.14).
Storage conditions: In a refrigerator protected from light.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group,
avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal
copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.

Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes
and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted once prior to start of the study and weekly (each formulation) during the treatment phase, according to a validated method. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42-44, 53 and 56 days).

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on a 28-day toxicity study in which 62.5, 250 and 1000 mg/kg were tested.
- Rationale for animal assignment: random

Positive control:

not needed

Examinations

Parental animals: Observations and examinations:

Mortality / Viability:
At least twice daily. The circumstances of any death were recorded in detail.

Clinical signs:
At least once daily from start of treatment onwards. The time of onset, grade and duration of any observed signs was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

Body weights:
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and
20 post-coitum and during lactation on Days 1 and 4.

Food consumption:
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 postcoitum and on Days 1 and 4 of lactation.

Water consumption:
Subjective appraisal was maintained during the study period. No treatment related effect was suspected.

General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
Deficiencies in maternal care, such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding, were recorded, if applicable.

Litter observations:

Mortality / Viability:
The numbers of live and dead pups on Day 1 of lactation and daily thereafter was determined. If possible, defects or the cause of death was evaluated.

Clinical signs:
At least once daily, detailed clinical observations were made for all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex:
Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

Animals surviving to scheduled necropsy were deeply anaesthetized using an isoflurane combination and subsequently exsanguinated.

Necropsy were conducted on the following days:
Females which deliver: Lactation Days 5-7.
Females which fail to deliver: Post-coitum Day 27 (females with evidence of mating).
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
Spontaneous deaths (Female no. 76): As soon as possible after death and always within 24 hours. Recognizable foetuses were examined externally, sexed (if possible), and euthanized by decapitation (if necessary)

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The number of former implantation sites and corpora lutea was recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin:
Cervix
Clitoral gland
Coagulation gland
Epididymides
Liver Testes
Mammary gland area (only required for females with total litter loss in which no pups of the litter have milk visible in the stomach)
Ovaries
Preputial gland
Prostate gland
Seminal vesicles
Stomach
Uterus
Vagina
Identification marks: not processed All gross lesions

Organ weights:
The following organ weights and terminal body weight were recorded from all F0-males on the scheduled day of necropsy:
Epididymides
Testes

Histopathology
The following slides were examined by a pathologist:
- The liver, stomach, ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4 to examine staging of spermatogenesis.
- The preserved organs and tissues of female no. 76 which died spontaneously.
- All gross lesions of all animals (all dose groups).
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate
gland, seminal vesicles, testes, uterus, and vagina) of male nos. 19 and 36 that failed to sire and female no. 59 that failed to deliver healthy pups.
On detection of possible treatment-related changes in the liver and stomach of any animal in the high dose group the histological examination was extended to these particular organs of the animals of Groups 2 and 3 (stomach: males and females, liver: males only). All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation between Days 5-7 of lactation.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 were accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may have displayed the same printed means for a given parameter, yet displayed different test statistics values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

CLINICAL SIGNS (PARENTAL ANIMALS)
There were no toxicologically relevant clinical signs noted up to 1000 mg/kg.

Salivation was noted for animals of all treated groups, however, this was considered to be due to the taste/irritancy of the test substance, and was not considered to be toxicologically relevant. Salivation was also noted on one occasion for a single control female; this was incidental in nature.

Other incidental findings included rales, which was seen for one animal each at 100, 300, and 1000 mg/kg, and piloerection that was seen for one female each at 300 and 1000 mg/kg, and scabs on the cheek. Lethargy, flat posture and ptosis were seen for one female at 100 mg/kg, however, due to the slight nature of the effects and their limited incidence, these signs were also considered to be incidental in nature, and thus not toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death, with female no. 76 (1000 mg/kg) found dead on day 19 of the treatment period. At macroscopic examination, this female was found with beginning autolysis and greenish fluid in the uterus. The cause of mortality could not be determined.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on body weights or body weight gains were seen with treatment up to 1000 mg/kg.

At 1000 mg/kg, body weight gains were significantly higher for females from Days 4-7 and Day 17 of the post coitum period. This was not considered to be toxicologically relevant because the differences from controls were relatively slight and reduced body weight gain or weight loss would be expected if treatment related toxicity were evident.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant effects on absolute or relative food consumption were seen with Ebecryl® 4858 treatment up to 1000 mg/kg.

Absolute food consumption was significantly higher for females over Days 1-8 of the premating period at 100 and 1000 mg/kg, and relative food consumption was significantly higher for females over Days 1-8 of the premating period at 300 and 1000 mg/kg, respectively. Males at 1000 mg/kg also had higher food consumption over Days 8-15 of the premating period. The increases compared to control values were only very slight, and were thus not considered to be toxicologically relevant.

Food efficiency:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic findings were noted in the stomach of males of Groups 3 and 4 and in the liver of males of Group 4.
Stomach:
- Hyperplasia of the forestomach / limiting ridge was noted in 5/10 males of Group 3 (minimal) and 5/10 males of Group 4 (4/10: minimal, 1/10: slight). This was also seen in 1/10 males of Groups 1 and 2 (100 mg/kg) at a minimal degree
- Lymphogranulocytic inflammation of the forestomach and/or limiting ridge was recorded at minimal degree in 4/10 Group 4 males. Minimal lymphogranulocytic inflammation of the stomach in the other Groups ranged from 1/10 in Groups 1 and 2 and 2/10 in Group 3. In these groups the inflammation was only located around the limiting ridge.
In females there were 2/10 Group 4 rats with a minimal degree of hyperplasia of the forestomach compared to 1/10 in Group 1. Since there were no gross lesions in the female stomach, this very minimal increase in incidence of hyperplasia of the forestomach in females was considered to be within background ranges, and thus not toxicologically relevant.
Liver:
- Hepatocellular hypertrophy was noted at minimal degree in 2/10 Group 4 males. While this constitutes a treatment related finding, it most likely represents an adaptive response and thus was not considered to be adverse or toxicologically relevant.
The remainder of the recorded microscopic findings were within the normal range of background alterations encountered in Wistar (Han) rats of this age and strain.
No abnormalities were seen in the reproductive organs of the males and females of Groups 2 (animal nos: 19 and 59) and 4 (animal nos: 36 and 76) that failed to mate, conceive, sire or deliver healthy pups, which could account for their infertility.
The spermatogenic staging profiles were normal for all Group 1 and Group 4 males.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

No toxicologically relevant effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic adverse effects were observed up to the highest dose

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicologically relevant effects on reproductive parameters were observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Small size was the only clinical symptom noted. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

No pups of the control group, one pup at 200 mg/kg and two pups at 300 mg/kg were missing, and one pup at 1000 mg/kg was found dead during the first days of lactation. Missing pups 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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were not affected by treatment.

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Description (incidence and severity):

Small size was the only macroscopic finding noted, and it was seen for only a single pup at 1000 mg/kg. This was incidental and was not attributable to treatment with the test substance. There were no macroscopic findings noted for the single pup that was found dead at 1000 mg/kg.

Details on results (F1)

No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Gestation
The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg.

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

Early postnatal pup development
The numbers of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects on early postnatal pup development

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the following (NOAEL) were determined: Parental (Systemic) NOAEL: 1000 mg/kg bw/day, Reproductive NOAEL: 1000 mg/kg bw/day, Developmental NOAEL: 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the reproductive-developmental toxicity of the test substance according to OECD Guideline 421 (reproduction/developmental toxicity screening study). Treatment with the test substance by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/d revealed local effects in males receiving 300 and 1000 mg/kg bw/d characterized by macroscopic and microscopic findings in the stomach (males only), which can be attributed to the irritating nature of the test compound. However, these effects were localized to the stomach, and there were no signs in any parameter examined that reflected systemic toxicity. The local effects are considered not to be relevant for human exposure and are attributed to high local exposure due to gavage. No toxicologically relevant effect on reproductive parameters was noted. The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment up to 1000 mg/kg bw/day. Further, no toxicologically relevant effect on the early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed. Under the study conditions, the parental systemic, reproductive and developmental NOAEL of the test substance in rats was determined to be 1000 mg/kg bw/d (Zmarowski, 2011).