Reaction mass of N, N'-hexane-1,6-diylbis [12-hydroxyoctadecanamide] and 12-hydroxy-N-[6-[1-oxoalkyl)amino] hexyl ] octadecanamide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2009 to 11 August 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction/Developmental toxicity screening test) and EPA OPPTS 870.3550 (Reproduction/Developmental toxicity screening test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): AD-1000
- Substance type: White powder
- Physical state: solid
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Males and females
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 11-12 weeks
- Weight at study initiation: males: 315-345 grams
- Fasting period before study: no
- Housing:
Pre-mating During acclimatization, females were housed in groups of 5 animals/cage in Macrolon cages (MIV type, height 18 cm).
Mating Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-coitum Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation Offspring was kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General Sterilized sawdust as bedding material and paper as cage-enrichment was supplied.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.9 -21.7°C
- Humidity (%): 42- 88 %
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 June 2009 To: 11 August 2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (factor 1.036).


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and based on a previous 28-day study with AD-1000 (NOTOX Project 455917).
- Concentration in vehicle: 10, 30 and 200 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:

Analyses were performed according to a validated method (NOTOX Project 456007).

Group 1-4 formulations were analysed for accuracy. Group 2 and 4 formulations were also analysed for stability (over 6 hours) and homogeneity.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. 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%.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: not specified in terms of days, but until successful pairing
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: not applicable
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 40 to 48 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 3 days of lactation.

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 150, 1000 mg/kg bw/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 selected based on a 28-day study with AD-1000 (NOTOX Project 455917).

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (early morning/late afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from Day 0 post-coitum onwards

BODY WEIGHT: Yes
- Time schedule for examinations: 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: Yes
- Food consumption for each animal determined: Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Reproduction processes: Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.

SACRIFICE
- Male animals: All surviving animals after 28 days of exposure
- Maternal animals: All surviving animals after at least 3 days of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- The number of corpora lutea and former implantation sites were recorded. Nongravid uteri were stained using the Salewski technique.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin:
Identification marks (not processed), Preputial gland, Cervix, Prostate gland, Clitoral gland, Seminal vesicles, Coagulation gland, Testes*, Epididymides*, Uterus, Ovaries, Vagina, Pituitary gland, All gross lesions
* Fixed in modified Davidson's solution and transferred to formalin after fixation for at least 24 hours.

ORGAN WEIGHTS
Epididymides
Testes

HISTOPATHOLOGY:
The following slides were examined by a pathologist:
- The ovaries and epididymides of the animals of Groups 1 and 4 and testes of all groups.
- The additional slides of the testes of the males of Groups 1 and 4 to examine staging of spermatogenesis.
- The reproductive organs (cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina) of all animals that failed to mate, conceive, sire or deliver healthy offspring.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously.
- All gross lesions of all animals (all dose groups).

Ovaries and uterine content:

See maternal examinations.

Fetal examinations:

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities,
GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

SACRIFICE
- Animals were subjected to external postmortem examinations.

GROSS NECROPSY
The stomach was examined for the presence of milk. Descriptions of all external abnormalities were recorded.
If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examination.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher 1950) was applied to frequency data.
- The corpora lutea and implantation sites were subjected to the Kruskal-Wallis onparametric ANOVA test (Kruskal, 1952) to determine intergroup difference. As no results of the ANOVA were significant (p<0.05), the Wilcoxon test (Wilcoxon, 1945) was not applied to the data.

All tests were two-sided and in all cases p < 0.05 was 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 be rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.

Indices:

For each group the following calculations were performed:

Percentage mating males = (Number of females mated/Number of males paired) x 100
Percentage mating females = (Number of females mated/Number of females paired) x 100
Fertility index males = (Number of males generating a pregnancy/Number of males paired) x 100
Fertility index females = (Number of pregnant females/Number of females paired) x 100
Conception rate = (Number of pregnant females/Number of females mated)x 100
Gestation index = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Percentage live males at first litter check: Number of live male pups at First Litter Check x 100/Number of live pups at First Litter Check
Percentage live females at first litter check: Number of live female pups at First Litter Check x 100/Number of live pups at First Litter Check
Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation x 100/Number of live pups at First Litter Check
Viability index: Number of live pups on Day 4 of lactation x 100/Number of pups born alive

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No toxicological relevant effects.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No toxicological relevant effects.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

At 1000 mg/kg (Group 4), postnatal loss was significantly increased to 10.9% of living pups lost from Days 0-4 post-partum compared to no pups lost in the control Group. In addition, there was a corresponding reduction in the viability index for Group 4, with a viability index of 89.1% compared to 100% for controls. As the postnatal loss was driven exclusively by one litter (female no. 71) that lost 16 pups over Days 0-4 post partum, it is considered not to reflect treatment-related toxicity.

In absence of a dose response relationship, no relevance was attached to the changed sex ratio observed at 150 mg/kg (sex ratio males/females was 60/40% in group 3 compared to 42/58% in the control group).

One pup of the control group, two pups of the mid dose group and sixteen pups of high dose group were found dead or missing during the first days of lactation. The increased number of pups found dead or missing in Group 4 was driven exclusively by one litter (female no. 71) that lost all but one pup over Days 0-4 post partum. As only one litter was affected, it is considered not to reflect treatment-related toxicity

Incidental clinical symptoms consisted of small and/or pale appearance, scabs on the nose and missing tail. No relationship with treatment was established for these observations and/or they were considered to be within the normal biological variation for rats of this age and strain.

Incidental macroscopic findings comprised small appearance, no milk in the stomach, missing tail and scabs on the nose. No relationship with treatment was established for these observations and/or they were considered to be within the normal biological variation for rats of this age and strain.

Macroscopic findings of pups that were found dead at the first litter check or in the days prior to scheduled necropsy included small size, cold, absence of milk in the stomach and/or autolysis. Cannibalism and stomach not present was noted for three of the sixteen pups that were lost from litter no. 71 (Group 4, 1000 mg/kg). No relationship with treatment was established for these findings.

Applicant's summary and conclusion

Conclusions:

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.
There were no treatment-related changes for mortality, clinical signs, body weight, food consumption or organ weights. Furthermore, macroscopic and microscopic examination did not reveal any findings that were related to treatment with AD-1000 up to 1000 mg/kg.
There were no treatment-related changes for reproduction, breeding data and pup development up to 1000 mg/kg/day. At 1000 mg/kg, increased postnatal loss and reduced viability index was noted. However, as this effect was driven exclusively by one litter (female no. 71) that lost all but one pup over Days 0-4 post partum, it is considered not to reflect treatment-related toxicity.

CONCLUSION
In conclusion, treatment with AD-1000 by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 1000 mg/kg body weight/day revealed no parental, reproduction, breeding and developmental toxicity up to 1000 mg/kg body weight/day.
The parental, reproduction, breeding and developmental NOAEL was established as being at least 1000 mg/kg body weight/day.