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Effects on fertility

Description of key information

Based on the available information, no adverse findings regarding reproduction were observed for the test item in an OECD 422 guideline study with rats. The NOAEL was determined to be ≥ 600 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Apr - 28 Nov 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
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
Version / remarks:
July 2000
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- 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)
Species:
rat
Strain:
Wistar
Remarks:
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
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
Dose / conc.:
43 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
Group 4
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.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS
- 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
- 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
- Tissues from all parental males (preserved in neutral phosphate buffered 4% formaldehyde solution): Prostate, Seminal vesicles with coagulating gland, Testes (in Bouin’s fixative), Epididymides (in Bouin’s fixative)
- Tissues from all parental females (preserved in neutral phosphate buffered 4% formaldehyde solution): Ovaries
- Tissues (preserved in neutral phosphate buffered 4% formaldehyde solution) from the five males and females per group selected for organ weights: 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
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in group 2 died accidentally after blood sampling. This death was not considered to be test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 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 and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
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
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Dose descriptor:
NOEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects on reproductive parameters observed
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
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 and weight changes:
no effects observed
Description (incidence and severity):
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.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
No abnormal findings were noted at macroscopic examination of the pups.
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOEL
Generation:
F1
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Critical effects observed:
no
Reproductive effects observed:
no
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.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive toxicity

Read-across justification for target substance (CAS14960-06-6):

The test item was assessed using the read-across approach. Since there are no studies for toxicity to reproduction for the test item, the read-across approach was followed using the structurally analogue substance (CAS 3655-00-3) as source substance. The substances strongly correlate because they have the same basic structure. The only difference is that they have a variance in the amount of sodium. As it is assumed that the toxicological effects are not primary triggered by the sodium content and the anion is the same for both substances, the Read-Across approach is suitable. See for additional information the RA justification attached to section 13.

 

Source study record (CAS 3655-00-3)

Valid information of the toxicological effects resulting from repeated oral-gavage administration of the source substance (CAS 3655-00-3) to rats, according to OECD guideline 422 are available (Harlan Laboratories Ltd., 2008). Therefore the source substance was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. The following dosage levels were administered: Group 1: 0 mg/kg bwt/day (control group), Group 2: 43 mg/kg bw/day, Group 3: 160 mg/kg bwday, Group 4: 600 mg/kg bw/day.

Dose levels were adjusted for content of the active ingredient (30.89%) and its purity (87%), applying a correction factor of 3.72. A standard dosage volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water). The following results were obtained: The NOAEL for systemic toxicity was determined to be 160 mg/kg bw/day. Results on repeated dose toxicity via the oral route showed no effects on reproduction or reproductive organs in parental animals. The litter size and mean number of pups at first litter check and on day 4 post partum were not affected by the treatment with the test item. Further, the sex ratio was not affected. Mean pup weight gain was not affected by treatment with the test item. At necropsy of pups, there were no abnormal findings. In absence of any altered parameters, the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 600 mg/kg bw/day, the highest dose tested.

 

Assessment of toxicity on reproduction

No adverse findings regarding reproduction (e.g. gonadal function, mating behavior, conception, development of the conceptus and parturition etc.) were observed in a combined subacute toxicity / reproduction screening study (OECD 422). Therefore the NOAEL for development and reproduction was established at the highest tested dose (600mg/kg bw/day).

In summary results from a combined subacute toxicity / reproduction screening study did not reveal any reason of concern for offspring and for parent animals with regard to developmental toxicity or fertility up to and including the limit dose (600mg/kg bw/day). Since significant scientific evidence for a lack of reproduction toxic effects of the substance is drawn from these results and an additional two generation study or a teratogenicity study at these concentration, is not expected to add any further relevant knowledge on this endpoint. No additional study is therefore propose due to animal welfare aspects.

Effects on developmental toxicity

Description of key information

Based on the available information, no adverse findings regarding developmental toxicity were observed for the test item in an OECD 422 guideline study performed with rat. The NOAEL was determined to be ≥ 600 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to chapter 13 for the read-across justification.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no effects on reproductive parameters
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed as far as F1 can be assessed in a screening study
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There are no valid, reliable and relevant studies available assessing the effects on developmental toxicity of the test substance.

Nevertheless as no maleformation or other adverse effects on offspring are reported in a OECD 422 study (Harlan, 2008) up to the highest test dosage (600mg/kg bw/day) no additional information is expected if a teratogenicity test is performed at that concentration. According to Annex XI REGULATION (EC) No 1907/2006 chapter 1.2 "weigh of evidence .... Where sufficient weight of evidence for the presence or absence of a particular dangerous property is available:.....—further testing on vertebrate animals for that property shall be omitted...." further testing (two generation or teratogenicity study) is not strongly suggested and therefore not proposed.

No adverse findings regarding reproduction (e.g. gonadal function, mating behavior, conception, development of the conceptus and parturition etc.) were observed in a combined subacute toxicity / reproduction screening study (OECD 422). Therefore the NOAEL for development and reproduction was established at the highest tested dose (600mg/kg bw/day)

In summary results from a combined subacute toxicity / reproduction screening study did not reveal any reason of concern for offspring and for parent animals with regard to developmental toxicity or fertility up to and including the limit dose (600mg/kg bw/day). Since significant scientific evidence for a lack of reproduction toxic effects of the substance is drawn from these results and an additional two generation study or a teratogenicity study at these concentration, is not expected to add any further relevant knowledge on this endpoint. No additional study is therefore propose due to animal welfare aspects.

Justification for classification or non-classification

Based on the available information classification for toxicity to reproduction or developmental toxicity is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Additional information