Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
22 Apr - 28 Nov 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. The reliability was set to 2 due to read-across.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22. March 1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650, July 2000
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium N-(2-carboxyethyl)-N-dodecyl-β-alaninate
EC Number:
222-899-0
EC Name:
Disodium N-(2-carboxyethyl)-N-dodecyl-β-alaninate
Cas Number:
3655-00-3
Molecular formula:
C18H35NO4.2Na
IUPAC Name:
disodium N-(2-carboxyethyl)-N-dodecyl-beta-alaninate
Details on test material:
- Name of test material (as cited in study report): Sodium coco β-iminodipropionate
- Physical state: liquid
- Analytical purity: 87%
- Active content: 30.89%
- Lot/batch No.: 184
- Expiration date of the lot/batch: 30. June 2008
- Storage condition of test material: room temperature (20 ± 5°C)

Test animals

Species:
rat
Strain:
other: HanRcc: WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories Ltd., Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: mean: males 294 - 344 g; females 176 - 221 g
- Housing: individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: Pelleted standard Kliba Nafag 3433 rat/mouse maintenance diet, ad libitum
- Water: community tap-water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
highly purified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosage formulations were prepared weekly prior to administration using the test item as supplied by the Sponsor and using a factor of 3.72 taking in account the purity of 87% and the content of the active ingredient of 30.89% (CAS 3655-00-3). Sodium coco β-iminodipropionate (CAS 3655-00-3) was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear, copulation plug observed referred to as day 0 post coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Several application formulations were prepared and representative analytical samples were collected and dispatched to the analytical laboratories internally. The test item concentrations were determined by HPLC coupled to an ELSD detector and quantified with the area under the peak.
Duration of treatment / exposure:
Males: minimum of 4 weeks (2 weeks prior to mating and 2 weeks of mating)
Females: approximately 7 weeks (2 weeks prior to mating up to day 4 post partum)
Frequency of treatment:
daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
43 (group 2), 160 (group 3), 600 (group 4) mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels were selected based on a previous dose range finding toxicity study in Han Wistar Rats.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- was recorded for male animals weekly during pre-pairing, for females weekly in during the pre-pairing period, gestation days 0-7, 7-14 and 14-21 post coitum, and days 1-4 post partum; food consumption was not recorded during the pairing period
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for any gross anomalies
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after at least 28 days of dosing
- Maternal animals: All surviving animals, on day 5 post partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites
- the following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative)
- the following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
Ovaries
- In addition, from the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions, Brain (cerebrum, cerebellum, pons), Spinal chord, Small and large intestines (incl. Peyer’s patches), Stomach, Liver, Kidneys, Adrenals, Spleen, Heart, Thymus, Thyroid and Parathyroid, Trachea and lungs (preserved by inflation with fixative and then immersion), Uterus (with vagina), Urinary bladder, Lymph nodes (mandibular and mesenteric), Peripheral nerve (sciatic), Bone marrow
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at day 5 post partum
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: examined macroscopically for any structural changes

Statistics:
The following statistical methods may be used to food consumption, body weight, macroscopic findings, organ weights and reproduction data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopic findings.
Offspring viability indices:
birth index = number of pups born alive/number of implantations

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
One female in group 2 died accidentally after blood sampling. This death was not considered to be test item-related.
In group 4, all animals were noted to push the head through the bedding material starting on day 9 of the pre-pairing period onwards. One female had salivation after administration of the test item on day 9 of the gestation period. These signs are considered to be signs of discomfort and not to be adverse. In groups 2 and 3, no clinical signs or observations were noted.

BODY WEIGHT AND BODY WEIGHT GAIN
- Males - pre-pairing period: In group 4, mean body weight gain was statistically significany decreased during the pre-pairing period (+6.6% versus +13.7% in the control group). Although this decrease had no statistically significant impact on the mean body weight, it was considered to be a test item related effect. During the pairing period, mean body weight and mean body weight gain were not affected by treatment with the test item. In groups 2 and 3, mean body weight and mean body weight gain were not affected by the treatment with the test item for the entire duration of the study. In group 3, mean body weight gain was statistically significant lower on day 6 of the pre-pairing period however, statistical significance occurred on a single day only and therefore it was not considered to be an adverse effect. During the pairing period, mean body weight gain was lower (+8.2% versus +12.0% in the control group) attaining statistically significance on days 9, 13 and 14. However, this was considered to be incidental as there was no-dose dependency.
- Females - pre-pairing, gestation and lactation periods: In group 4, mean body weight gain was statistically significant decreased during the pre-pairing period (+3.5% versus +8.2% in the control group). This was considered to be a test item related effect, although it did not affect the mean body weight. During the gestation and lactation periods, mean body weight and mean body weight gain were not affected by treatment with the test item. In group 3, mean body weight gain was lower during the pre-pairing period (+5.0% versus +8.2% in the control group) attaining statistical significance between day 6 and 8 and on days 11 and 14. This was considered an effect of the test item although mean body weight was not affected. During the gestation and lactation periods, mean body weight and mean body weight gain were not affected by treatment with the test item. In group 2, mean body weight and mean body weight gain were not affected for the entire duration of the treatment with the test item.

FOOD CONSUMPTION
- Males - pre-pairing period: In group 4, mean food consumption was statistically significant reduced between days 1-8 of the pre-pairing period (-19.2% compared to the control group) and it remained lower between days 8-14 (-10.6% compared to the control group) without attaining statistical significance. In group 3, mean food consumption was not considered to be affected by the treatment with the test item. As mean food consumption was slightly lower between days 1 and 8 of the pre-pairing period (-4.9% compared to the control group) and slightly higher between days 8 and 14 of the pre-pairing period (+2.1% compared to the control group), these variations were considered to be incidental. In group 2, no test item-related effects were noted.
- Females - pre-pairing, gestation and lactation periods: In group 4, mean food consumption was statistically significant decreased between days 1-8 of the pre-pairing period (-15.6% compared to the control group). This was considered to be a test item-related effect. During the gestation and lactation periods mean food consumption was not considered to be affected by treatment with the test item. The statistically significant higher food consumption during the lactation period (+23.0% compared to the control group) was considered to be incidental. In group 3, mean food consumption was decreased between days 1-8 of the pre-pairing period (-10.8% compared to the control group). This was considered to be a transient test item-related effect even thought it was not statistically significant. During the gestation and lactation periods, no test item-related effects were noted. In group 2, mean food consumption was not affected by treatment with the test item. During the lactation period, the statistically significant higher mean food consumption was considered to be incidental.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
All females mated within the first pairing period. The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 4.6, 3.5, 5.3 and 4.2 days in groups 1, 2, 3 and 4, respectively. The median precoital time was 3, 3, 6 and 3 days in order of ascending dose level. The higher median precoital time noted in group 3 was considered to be incidental since there was no dose dependency.
The mean duration of gestation was unaffected by exposure to the test item. Mean duration of gestation was 21.3, 21.6, 20.7 and 21.3 days, in order of ascending dose level.
The mean number of implantations per litter and mean incidence of post-implantation loss as a percentage of total implantations were not affected by the treatment with the test item. Mean number of implantation sites was 12.9, 13.8, 13.6 and 13.4 and mean incidence of postimplantation loss was 12.1, 10.9, 7.4 and 6.6% in groups 1, 2, 3 and 4, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no changes in organ weights for organs of the reproductive system.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No gross anomalies were detected.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological evaluation of the reproductive organs did not reveal any relevant changes in high-dose animals. Special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure did not reveal any differences between control (group 1) and high-dose (group 4) males.

Effect levels (P0)

Dose descriptor:
NOEL
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproductive parameters observed

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The birth index (number of pups born alive/number of implantations) was not affected by treatment with the test item. The birth index was 87.9, 89.1, 92.6 and 93.4% in groups 1, 2, 3 and 4, respectively. Mean litter size at first litter check was 11.3 12.3, 12.6 and 12.6 in groups 1, 2, 3 and 4, respectively. Mean postnatal loss was 0.1, 0.0, 0.2 and 0.0 in groups 1, 2, 3 and 4, respectively. Correspondingly, the viability index was 99.0, 100.0, 98.2 and 100.0%.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 1 post partum and during the lactation period (to day 4 post partum) were unaffected by treatment with the test item. On day 1 post partum mean pup weights were 5.9, 6.2, 5.9 and 5.7 g in order of ascending dose level. Mean pup weights on day 4 post partum were 8.4, 8.9, 8.2 and 8.0 g in order of ascending dose level.

SEXUAL MATURATION (OFFSPRING)
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. In group 3, the statistically significant lower number of females was considered to be incidental. The proportion of males was 45, 44, 58 and 50%, in order of ascending dose level.

GROSS PATHOLOGY (OFFSPRING)
No abnormal findings were noted at macroscopic examination of F1 pups.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: as far as F1 can be assessed in a screening study

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In absence of any altered parameters, the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 600 mg/kg/day, the highest dose tested.