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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

Qualifier:

according to guideline

Guideline:

other: OECD 421: Reproduction/Developmental Toxicity Screening Study

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

(specific gravity 1.125)

Details on oral 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.

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

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

not needed

Examinations

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death, with female (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:

effects observed, non-treatment-related

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):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on absolute or relative food consumption were seen with the test substance 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

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights and organ to body weight ratios were unaffected by treatment up to 1000 mg/kg.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related findings were noted in the stomach of males. At 300 mg/kg and 1000 mg/kg, the number of animals with a thickened limiting ridge of the stomach noted at necropsy was significantly higher than controls. There were 5/10 and 7/10 males noted with this in the 300 and 1000 mg/kg groups, respectively.
Two males at 300 mg/kg were noted with reddish foci on the stomach glandular mucosa, and a single male at 1000 mg/kg was noted with irregular surface of the limiting ridge of the stomach. Because a significant increase in stomach findings for males in these groups were seen, a relationship to treatment cannot be excluded, despite the limited incidence seen.

Neuropathological findings:

not specified

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.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
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

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic effects observed up to the high dose level

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the study conditions, Parental (Systemic) NOAEL was established at 1000 mg/kg/day.

Executive summary:

A study was conducted to determine the short term repeat dose toxicity of the test substance in rats according to OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Study). Crl:WI(Han) (outbred, SPF-Quality) rats were tested in this study. Males were exposed for 2 weeks prior to mating, during mating, and up to termination. The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 d of lactation. The test substance was administered at 0, 100, 300 and 1000 mg/kg bw/d once daily for 7 d per week. There were no adverse effects on clinical signs, body weight, food consumption and organ weights at necropsy up to the highest tested dose. Macroscopic findings were limited to thickened limiting ridge of the stomach and reddish foci on the stomach glandular mucosa in male rats at 300 and 1000 mg/kg bw/d. Microscopically, hyperplasia and lymphogranulocytic inflammation of the forestomach were observed in the affected males which correlates well with the local irritation effects of repeated dose oral gavage of the test substance. Under the study lconditions, the parental NOAEL for systemic effects were determined to be 1000 mg/kg/day (Zmarowski, 2011).