Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from similar mixture/product

Adequacy of study:

key study

Study period:

April 2008 - December 2008

Reliability:

1 (reliable without restriction)

Justification for type of information:

Read-across justification is given in separate supportive texts in section headers and section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The purpose of this study was to generate information concerning the effects of Sodium coco βiminodipropionate (CAS # 3655-00-3) on the possible health hazards likely to arise from
repeated exposure over a relatively limited period of time. In addition, it provides information on
possible effects on male and female reproductive performance such as gonadal function, mating
behavior, conception, development of the conceptus and parturition.

Test material

Test material information

Constituent 1

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

IUPAC Name:

disodium N-(2-carboxyethyl)-N-dodecyl-beta-alaninate

Test material form:

liquid

Details on test material:

Active content: 30.89%
Purity: 87%

Specific details on test material used for the study:

Expiry date 30 June 2008

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a
recommended test system.
Breeder: Harlan Laboratories Ltd.
Laboratory Animal Services
Wölferstrasse 4
4414 Füllinsdorf / Switzerland

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of Animals: 40 males: 10 per group
40 females: 10 per group*
Age (at Start of Treatment): 11 weeks
Body Weight Range
(at Start of Treatment):
Males: 294 to 344 g
Females: 176 to 221 g
Identification: Cage card and individual animal number (ear tattoo).
Pups: On day 1 post partum, pups were individually
tattooed with Indian ink.
Randomization: Computer-generated random algorithm. In addition
body weights (recorded on the day of allocation)
were taken into consideration in order to ensure
similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only
animals without any visible signs of illness were
used for the study.

Husbandry
Room Number, Füllinsdorf: 011B
Conditions: Standard laboratory conditions. Air-conditioned with
10 - 15 air changes per hour, continuously monitored
environmental conditions (temp. range: 22 ± 3 °C;
relative humidity range: 30 - 70%). There was a
12-hour fluorescent light / 12-hour dark cycle with
music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire
mesh tops and sterilized standard softwood bedding
(‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
During the pre-pairing period, cages with males
were interspersed among those holding females to
promote the development of regular estrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 (batch no.77/07)
rat / mouse maintenance diet (Provimi Kliba SA,
4303 Kaiseraugst / Switzerland) was available ad
libitum.
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Treatment
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted
route of exposure for studies of this type.
Frequency of Administration: Daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group)
Group 2: 43 mg/kg/day
Group 3: 160 mg/kg/day
Group 4: 600 mg/kg/day
Rationale for Dose Level Selection: The dosage levels were selected based on a previous
dose range finding toxicity study in Han Wistar Rats,
(RCC Study Number B61108 non-GLP Study),
using dose levels of 100, 300 and 1000 mg/kg/day
and resulting in some effects on body weight and/or
food consumption in the second part of the study
noted also at 100 mg/kg/day.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males: Minimum 4 weeks
Females: Approximately 7 weeks

Details on mating procedure:

Mating, Gestation and Lactation
During the pairing period, females were housed with sexually mature males (1:1) in special
automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until
evidence of copulation was observed. This system reduced the variation in the copulation times
of the different females. The females were removed and housed individually if:
a) The daily vaginal smear was sperm positive, or
b) A copulation plug was observed.
The day of mating was designated day 0 post coitum.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum.
Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of Dose Formulations
On the first day of treatment samples from the control group as well as three samples (top,
middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of
concentration and homogeneity. Samples of about 2 g of each concentration were taken from the
middle only to confirm stability (4 hrs and 7 days). During the last week of the treatment,
samples were taken from the middle to confirm concentration. The aliquots for analysis of dose
formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan
Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.
The samples were analyzed following an analytical procedure developed at Harlan Laboratories.

Duration of treatment / exposure:

Duration of Treatment Period: Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Frequency of Administration: Daily

Details on study schedule:

See Table below

Males were sacrificed after treatment of at least 28 days, when no longer needed for assessment
of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on
day 5 post partum.
If birth did not occur on the expected date (approximately day 21 post coitum), the dam was
sacrificed on days 22-26 post coitum.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

43 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

160 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males per group
10 females per group

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Observations
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily,
during acclimatization and up to day of necropsy).
Additionally, females were observed for signs of
difficult or prolonged parturition, and behavioral
abnormalities in nesting and nursing.
Food Consumption: Males: Weekly during pre-pairing
Females: Pre-pairing period days 1-8 and 8-14;
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.
Body Weights: Recorded daily from treatment start to day of
necropsy.
Pup Data: The litters were examined for litter size, live births,
still births and any gross anomalies. The sex ratio of
the pups was recorded. Pups were weighed
individually (without identification) on days 0 (if
possible), 1 and 4 post partum.

Detailed Clinical Observations
Once prior to the first administration of the test item and weekly thereafter, detailed clinical
observations were performed outside the home cage. Animals were observed for the following:
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.

Functional Observation Battery
At one time during the study (males shortly before the scheduled sacrifice and females on day 3
or 4 post partum) relevant parameters were performed with five P generation males and five
P generation females randomly selected from each group. This FOB assessment was conducted
following the daily dose administration. Animals were observed for the following:
a) Cage-side observations: unusual body movements (e.g. tremors, convulsions),
abnormal behavior (e.g. circling, stereotypy) and posture as well as resistance to
removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil
reactivity, salivation, muscle tone, extensor thrust response, righting reflex and
reaction to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute
evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of
urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behavior,
respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or feces,
soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal
temperature.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was
measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based
on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and
the total activity over 30 minutes were reported.

Clinical Laboratory Investigations
Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males (randomly selected) from each group. Blood samples from 5 lactating females
(randomly selected) from each group were obtained on day 5 post partum. Blood samples were
drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted
for approximately 18 hours before blood sampling but allowed access to water ad libitum. The
samples were collected early in the working day to reduce biological variation caused by
circadian rhythms.
The assay was performed under the responsibility of R. Draheim at Harlan Laboratories Ltd.
(Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.

Hematology
The following hematology parameters were determined:
Complete Blood Cell Count
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Leukocyte count, total
Differential leukocyte count
Platelet count
Coagulation
Prothrombin time (= Thromboplastin time)
Activated partial Thromoplastin time

Clinical Biochemistry
The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

Oestrous cyclicity (parental animals):

During the pre-pairing period, cages with males
were interspersed among those holding females to
promote the development of regular estrus cycles.

Sperm parameters (parental animals):

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial
cell structure.

Litter observations:

The litters were examined for litter size, live births,
still births and any gross anomalies. The sex ratio of
the pups was recorded. Pups were weighed
individually (without identification) on days 0 (if
possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

Necropsy
All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77®). All P
generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at
the scheduled necropsy or during the study if death occurred.
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 testes and epididymides of all parental males were weighed as pairs.
In addition, from 5 males and females selected randomly from each group, the following organs
were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Liver
Thymus
Spleen

Tissue Preservation
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):

Necropsy
All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77®). All P
generation animals were exsanguinated.
Dead pups, except those excessively cannibalized, were examined macroscopically.
All pups were examined macroscopically for any structural changes, either at
the scheduled necropsy or during the study if death occurred.

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.

Reproductive indices:

For reproduction data, group mean values were calculated both on a litter basis and on a
percentage per group basis. Mean pup weights were calculated from the individual weights both
on a per group and on a per litter basis.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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.

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):

Body Weights - Males
Pre-pairing and Pairing Periods
In group 4, mean body weight gain was statistically significantly decreased during the prepairing 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 itemrelated 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 significantly 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.

Body Weights - Females
Pre-pairing, Pairing, Gestation and Lactation Periods
In group 4, mean body weight gain was statistically significantly decreased during the prepairing period (+3.5% versus +8.2% in the control group). This was considered to be a test itemrelated 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):

Food Consumption - Males
In group 4, mean food consumption was statistically significantly 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.

Food Consumption - Females
Pre-pairing, Gestation and Lactation Periods
In group 4, mean food consumption was statistically significantly 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 significantly 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 significantly higher mean food consumption was considered to
be incidental.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males
In group 4, the absolute count of neutrophils was statistically significantly higher (+40.6%
compared to the control group). Since the relative count was not statistically significantly
increased, this was not considered to be adverse. Mean corpuscular hemoglobin concentration
was slightly but statistically significantly decreased (-2.8% compared to the control group).
Since the mean corpuscular hemoglobin was not affected, it was not considered to be an adverse
effect.
In group 3, the statistically significantly higher relative red cell volume distribution width
(+19.0% compared to the control group) was within the range of historical reference value.
In group 2 no changes were noted.

Females
The assessment of the hematology data did not reveal any test item-related effects in females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males
In group 4, urea and potassium concentrations were statistically significantly increased (+25.0%
and +15.2%, respectively, compared to the control group). These alterations correlate with the
histopathological findings noted in the kidney, thus they were considered to be test item-related.
In group 3, the statistically significantly lower phosphorus concentration (-13.6% compared to
the control group) was within the range of historical reference values. The statistically
significantly lower concentration of total bilirubin in groups 2 and 3 (-27.6% and -26.8%
compared to the control group, respectively) was not considered to be a test item-related effect
because there was no dose-dependency.
Females
The assessment of the clinical biochemistry parameters did not reveal any test item-related effect.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery
There was no indication of any test item-related effect during the open field phase.
Mean values of grip strength (fore- and hind paws) and landing foot splay gave no indication of
any test item-related effects.
In groups 3 and 4, mean body temperature was statistically significantly reduced in males
compared to the control group (37.5 °C and 37.4°C, respectively, compared to 38.6 °C in the
control group).

Locomotor Activity
Locomotor activity was assessed quantitatively in terms of low beam counts in an activity
monitor. In all groups there was no indication of a test item-related effect. The statistically
significantly lower locomotor activity noted at 30 min in group 4 males, and the statistically
significantly lower total locomotor activity noted in group 4 females were within the range of the
historical control data.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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.
Liver
Minimal centrilobular hepatocellular hypertrophy was observed in four males in group 3 and
slight centrilobular hepatocellular hypertrophy in one male in group 4; minimal to slight diffuse
hepatocellular hypertrophy was noted in the remaining four males and all five females in group 4.
Both types of liver cell hypertrophy were considered to represent an adaptive response to the
increased need for metabolizing the test item.
Thyroid gland
In group 4, minimally increased incidence and severity of diffuse follicular cell hypertrophy was
noted in males and females. This change was considered to be secondary to the enhanced liver
cell metabolism due to hepatocellular hypertrophy.
Stomach

Minimal acanthosis of nonglandular stomach was present in all males and in three females in
group 4 and was associated with minimal to slight focal/multifocal chronic inflammation in two
of these males. Isolated, slight and multifocal chronic inflammation occurred in two females in
group 3 and one male in group 4.
Minimal multifocal chronic inflammation in the glandular stomach was noted in one male in
group 3, and slight focal chronic inflammation in one female in group 3. The findings in the
female were associated with minimal and multifocal congestion. Slight multifocal congestion
occurred in two females in group 4, in one female associated with an acute thrombus and in the
other with minimal and multifocal chronic inflammation. All of the stomach findings were
considered to represent a local irritating effect of the test item.
Kidneys
Minimally increased incidence of hyaline droplets/granules was observed in males in groups 3
and 4 (five males in each group, compared to three in the control group). These eosinophilic
hyaline droplets/granules, which reflected an increased content of α-2µ-globulin, occur within
the cytoplasm of proximal convoluted tubules of sexually mature male rats. Because a slightly
increased severity was also noted (mean severity of 2.2 compared to 1.0 in the control males),
this was considered to be an adverse effect of the test item in group 4 males.
Spleen
Extramedullary hematopoiesis was minimally increased in severity in females in group 4,
possibly secondary to or an adaptation to stress and/or impaired health conditions.
In group 4, the minimally increased incidence of thymus atrophy in females was also considered
to be secondary to stress rather than to represent a direct effect of the test item.
All other microscopic findings noted in various organs and in all groups examined were considered to be incidental in nature because their morphology, severity, and incidence did not distinguish treated rats from controls

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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 dosedependency.

Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.4,
15.1, 14.8 and 15.2 in order of ascending dose level) and gave no indication of any test itemrelated effect.

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.

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%.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 43 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other: SD opinion within report

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal findings were noted at first litter check or during the first 4 days post partum.

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal findings were noted at macroscopic examination of F1 pups.

Other effects:

no effects observed

Description (incidence and severity):

Sex Ratios
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 significantly 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.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 600 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: SD opinion within report

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analysis of Dose Formulations

The dosage formulations investigated during the study were found to comprise Sodium coco ß-iminodipropionate (CAS # 3655-00-3) in the range of 92.1% to 104.0% and, thus, the required content limit of ±20% with reference to the nominal concentration was met. The homogeneous distribution of Sodium coco ß-iminodipropionate (CAS # 3655-00-3) in the preparations was approved because single results found did not deviate more than 4.9% (

Applicant's summary and conclusion

Conclusions:

No reproductive effects observed up to 600 mg/kg/day