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Toxicological information

Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 2-[methyloleoylamino]ethane-1-sulphonate
EC Number:
EC Name:
Sodium 2-[methyloleoylamino]ethane-1-sulphonate
Cas Number:
Molecular formula:
sodium 2-[methyl(oleoyl)amino]ethanesulfonate
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Physical state: Yellowish pale powder (Appearance at first use: Yellowish powder)
- Storage condition of test material: Room temperature, dry place

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River, S.r.l. Calco, (Lecco), Italy.
- Age at study initiation: 8-9 wks
- Weight at study initiation: (P) Males: 255.13-257.18 g; Females: 193.39-193.90 g
- Fasting period before study: no
- Housing: 5 per cage (polysulfone solid bottomed cages)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approximately 2 wks

- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): approximately 15-20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 20 June - 12 August 2012

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.

- Concentration in vehicle: 10, 30 and 100 mg/mL
Details on mating procedure:
- M/F ratio per cage: one to one
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): single
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 91450).
The validation of the formulation procedure for test substance was performed at 10, 30 and 100 mg/mL. The results were obtained in RTC Study
No. 91450 and all levels were within the acceptability limits for concentration and homogeneity.
The stability was found to be 24 hours at room temperature in the concentration range of 10 to 100 mg/mL in the same study.
Samples of the formulations prepared on Weeks 1 and 6 (when all females were present) of the present study were also analysed to check the concentration and homogeneity.
The overall results of the analyses were within the limits of acceptance stated in RTC’s SOPs for concentration (90-110%) and homogeneity (CV <10%).
The software used for this activity was Analyst 1.5.2 (AB Sciex).
Duration of treatment / exposure:
Males: about 28 days (Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.)
Females: about 50 days (Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, and thereafter during pairing, post coitum and post partum periods until Day 3 post partum.
Frequency of treatment:
Once a day
Details on study schedule:
Males were treated for a total of 33 days including 2 weeks prior to pairing and continuously thereafter up to the day before necropsy.
Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3
post partum.
Doses / concentrations
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
nominal in water
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages of 100, 300 and 1000 mg/kg/day were selected in consultation with the Sponsor based on preliminary dose-range-finder. The oral route was selected as it is a possible route of exposure of the test item in man.


Parental animals: Observations and examinations:
- Morality: throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and public holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
- Clinical signs : All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

Food consumption was recorded at weekly intervals by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 post partum).
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Sperm parameters (parental animals):
Parameters examined in all control and high dose parental male generations:
Epididymides, testis, prostate gland and seminal vesicles weights, morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle)
Litter observations:
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies. Live pups were individually weighed on Days 1 and 4 post partum. Observation was performed once daily for all litters.

yes, for external and internal abnormalities
Postmortem examinations (parental animals):
- Male animals: All surviving animals were killed after the mating of all females (i.e. after 33 days of treatment).
- Maternal animals: The females with live pups were killed on Day 4 post partum

The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted
(including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples
preserved in fixative and processed for histopathological examination.

All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).

The uterus of female no. 91480063 with no visible implantations was immersed in a 20% solution of ammonium sulphide to reveal evidence of

From all animals completing the scheduled test period the organs listed in Annex 1 of the study protocol were dissected free of fat and weighted. The ratios of organ weight to terminal body weight were calculated for each animal.

Tissues fixed and preserved
Samples of all the tissues listed in the Annex 1 of the study protocol were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid) from all animals.

Histopathological examination and staging of spermatogenic cycle
The tissues required for histopathological examination are listed in Annex 1 of the study protocol. After dehydration and embedding in paraffin wax,
sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:
a) Tissues from all animals in the control and high dose groups killed at term.
b) Tissues from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.

Postmortem examinations (offspring):
- Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopenthal.

- All pups found dead in the cage were examined for external and internal abnormalities. Sex was also confirmed by gonadal inspection.
- All live pups were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
The criterion for statistical significance was p<0.05.
Reproductive indices:
Fertility Index (%)=no. of males which induced pregnancy/no. of males paired x 100

Fertility Index (%) =no. of pregnant females/no. of females paired x 100

Males and females
Copulatory Index (%) =no. of animals mated/no. of animals paired x 100
Copulatory Interval = Mean number of days between pairing and mating

Offspring viability indices:
Pre-implantation loss was calculated as a percentage from the formula: (No. of corpora lutea - No. of implantations)/ No. of corpora lutea x 100
Pre-birth loss was calculated as a percentage from the formula: (No. of visible implantations - total litter size at birth )/ No. of visible implantations x 100
Pup loss at birth was calculated as a percentage from the formula: (Total litter size - live litter size)/ Total litter size x 100
Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula: (Total litter size at birth - live litter size at Day 4)/Total litter size at birth x 100
Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Two high dose animals were found dead during the study: one male on Day 8 of the study and one female on Day 0 post coitum. The cause of death was attributed to a mis-dosing on the basis of the macroscopic and histopathological findings. All females were found pregnant at necropsy with the exception of the found dead animal.

Salivation was observed in the majority of high dose animals of both sexes throughout the study and occasionally in the low and mid-dose groups. This sign appeared early after dosing.
In one occasion, difficulty in breathing (dyspnoea) was observed in two high dose males.
No other relevant signs were recorded.
In several occasions, soft faeces and red/brown staining in the cage tray were noted in treated groups during the mating phase.
These data were not tabulated but retained as study raw data.

Body weight and body weight gain were comparable between the groups throughout the study for both males and females.
Measurement of food consumption did not reveal relevant differences between the groups.

Measurements of oestrus cycle, pre-coital intervals, copulatory and fertility indices did not show differences between treated and control groups.

Implantation, pre-birth loss data and gestation length of females
No significant differences were found in the number of implantations, corpora lutea, total litter size, pre-implantation and pre-birth loss between control and treated groups.
Gestation length was comparable between groups. The number of females with live pups on Day 4 post partum was: 10 in each of the control, low and mid- dose groups and 9 in the high dose group.

Terminal body weight and organ weights
No differences were found in terminal body weight nor in organ weights between the groups.

Unscheduled deaths
A high dose male and female died during the experimental phase.
At post mortem examination, the most relevant changes detected in the male or female rat were: red or brown staining in the muzzle, incomplete collapse, red colour or multiple dark areas in the lungs or red fluid in the thoracic cavity or red colour in the cervical lymph nodes or in the thymus.
Such changes, also confirmed by histopathological examination, were considered related to a possible misdosing and as consequence the factor contributory to death.
Final sacrifice
No relevant changes were detected at post mortem examination in treated animals, when compared with controls.

No treatment-related changes were seen in selected organs/tissues evaluated in males or females receiving the test substance, sacrificed at the end of
treatment period, nor in the abnormalities detected in all groups at post mortem examination.
The sporadic lesions reported in single treated animals were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species and age under the experimental conditions.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Effect levels (P0)

Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOAEL corresponding to the highest dose tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Mean litter and pup weights were comparable between groups on Days 1 and 4 post partum.

No differences were noted in the clinical signs observed in pups between groups.

No differences were found in sex ratio.

No milk in stomach and autolysed organs in the abdomen were observed at necropsy in the decedent pups of control and treated groups.
No abnormalities were found in pups sacrificed on Day 4 post partum. Only one high dose pup had no milk in stomach.

Effect levels (F1)

Dose descriptor:
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOAEL corresponding to the highest dose tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity
could be considered ≥ 1000 mg/kg/day for both males and females.