Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
oct 2017 - july 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Purity: 100 % UVCB
Batch identification: 17000229U0
Physical state/appearance: Liquid, highly viscous / yellowish to brownish

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: supplied by Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks

- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%.
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours both

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations
were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with deionized water in a calibrated beaker and intensely mixed with a magnetic
stirrer until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0.15g/100 ml, 0,5g/100 ml and 2,0g/100 ml
- Amount of vehicle (if gavage): up to 9,85 ml see above
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany,
under the responsibility of the study director of this test facility (BASF Project No. 02Y0054/08X094).
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day
(GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the
experimental phase) and first administration (GD 6).

The body weight of the pregnant animals on day 0 varied between 143.0 – 192.3 g.
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always
at approximately the same time in the morning.
Frequency of treatment:
daily, GD6-GD19
Duration of test:
till GD 20
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
50 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on available study 85R0686/01x043

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs
of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

DETAILED CLINICAL OBSERVATIONS: Yes
see above

BODY WEIGHT: Yes
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.

FOOD CONSUMPTION: Yes
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No data


POST-MORTEM EXAMINATIONS: Yes

Pathology
On GD20 all surviving dams were sacrificed by decapitation under isoflurane anesthesia in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross
pathology, special attention being given to the reproductive organs and the gastrointestinal tract.
The following weights were determined in all animals sacrificed on schedule:
1. Adrenal glands
2. Liver
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
3. All gross lesions
4. Adrenal glands
5. Liver
6. Stomach

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Cesarean section
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.
After the dams had been sacrificed, they were necropsied and assessed for gross pathology
The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI
from uteri from apparently non-pregnant animals and the empty uterus horn in the
case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the
uterus had been opened)
Fetal examinations:
- External examinations: Yes
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia.
Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded.
Thereafter, the fetuses were sacrificed. After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.
- Soft tissue examinations: Yes
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR.
- Skeletal examinations: Yes
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL.
- Head examinations: No data
Statistics:
Statistical analyses were performed according to the following:

Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened
uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation
loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

with

Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means


Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

with

Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions


Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

with

Pairwise comparison of each dose group with the control group using the WILCOXONtest (one-sided) for the hypothesis of equal medians


Weight parameters

with

Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians
Indices:
The conception rate (in %), preimplantation loss (in %), postimplantation loss (in %) was calculated
Historical control data:
Historical control data of reproduction toxicology and Historical control data of pathology is given as supplement

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All females of the high-dose group (200 mg/kg bw/d) and five females of the mid-dose group (50 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred
in the respective animals only within the 2-hour examination interval (i.e. 0-2h after treatment) and was initially observed on GD 7 (200 mg/kg bw/d) or GD 19 (50 mg/kg bw/d).
Furthermore, nearly all (18 out of 25) females of the high-dose group (200 mg/kg bw/d) ploughed nose-first into bedding during the treatment period (within 0-2h after treatment). This
finding was initially observed on GD 14. Both findings are considered to be treatment-related, most likely as a local irritation of the upper digestive tract or as a result of the bad taste of the
test substance/vehicle preparation. They are not considered to be a sign of systemic toxicity.
During the 5-hour examination interval (i.e. >2<5h after treatment), no clinical signs or changes
of general behavior were detected in any female of all test groups.
No further clinical signs or changes of general behavior, which may be attributed to the test
substance, were detected in any female at dose levels of 15, 50 or 200 mg/kg bw/d during the
entire study period.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 15, 50 or 200 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (15, 50 and 200 mg/kg bw/d) were generally comparable to the concurrent control group
throughout the entire study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the dams in test group 3 (200 mg/kg bw/d) was statistically significantly reduced on GD 6-8, GD 10-13 and GD 19-20 (up to -10 % in comparison to the concurrent control). If calculated for the entire treatment (GD 6-19) period, the high-dose dams consumed about 4% less food than the controls. Although the decrease during GD 6-19 was not statistically significant, three treatment intervals showed a statistically significant decrease in food consumption. Overall, this was assessed as treatment-related and adverse.
The mean food consumption of the dams in test groups 1 and 2 (15 and 50 mg/kg bw/d) was comparable to the concurrent control group throughout the entire study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
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:
effects observed, treatment-related
Description (incidence and severity):
Uterus weight
The mean gravid uterus weights of the animals of test groups 1-3 (15, 50 and 200 mg/kg bw/d) were not influenced by the test substance.

Absolute organ weights
All mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly increased for adrenal gland and liver.
The minimally, but significantly increased relative liver weights in test group 3 animals were regarded as treatment-related. Both, absolute (+7%, not significant) and relative (+8%, significant)
liver weights were above the range of historical control values. The significantly decreased weights of adrenal glands in test group 1 animals as well as the significantly increased weights of adrenal glands in test group 3 animals were regarded as incidental, since there was no dose-response-relationship.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The thickened wall of the forestomach (25/25), glandular stomach (9/25) and duodenum (10/25) as well as the focus and the deposition in the glandular stomach (1/25 each) of test
group 3 animals were regarded as treatment-related.

The enlarged livers in 4/25 animals were regarded as treatment-related and correlated to slightly increased liver weights.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No differences of toxicological relevance between the control and the treated groups (15, 50 or 200 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate,
mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No differences of toxicological relevance between the control and the treated groups (15, 50 or 200 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate,
mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No differences of toxicological relevance between the control and the treated groups (15, 50 or 200 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate,
mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
not examined
Details on maternal toxic effects:
The combination of different individual findings (a reduction in food consumption, decrease in corrected body weight
gain, thickened wall in stomach and duodenum) at 200 mg/kg bw/d was assessed as adverse.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: The combination of different individual findings (a reduction in food consumption, decrease in corrected body weight gain, thickened wall in stomach and duodenum) was assessed as adverse.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (15, 50 and 200 mg/kg bw/d) was comparable to the control fetuses.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
No soft tissue malformations were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus in test group 3 (200 mg/kg bw/d) had a skeletal malformation (misshapen lumbar vertebra). This isolated finding occurred in one single fetus. No
ontogenetic pattern is recognizable for this individual malformation nor was there any cluster of this individual malformation seen in the other offspring of the test groups. Thus, no association
to the treatment was assumed. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal skeletal variations
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to
several parts of fetal skeletons and appeared in the majority of cases without a relation to dose.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups.
The overall incidences of skeletal variations were comparable to the historical control data.
Details on embryotoxic / teratogenic effects:
External and soft tissue malformations did not occur in any of the fetuses in this study. There was noted one skeletal malformation in test group 3 (200 mg/kg bw/d), i.e. misshapen
lumbar vertebra. This isolated finding occurred in one single fetus (94-08) which had additionally a lower body weight (2.5 g) compared to the group mean value (3.8 g). No ontogenetic
pattern is recognizable for this individual malformation nor was there any cluster of this individual malformation seen in the other offspring of the test groups. It also does neither form
a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study. Thus, there is no evidence for
any association of this finding with the treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no embryotoxic / teratogenic effects at highest concentration

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid dose of 50 mg/kg bw/d.
The NOAEL for prenatal developmental toxicity is the highest tested dose of 200 mg/kg bw/d.
Executive summary:

Sulfonium compounds, C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts) was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered

as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 15, 50 and 200 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (deionized water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group.The state of health of the animals was checked each day.

The following test substance-related adverse effects/findings were noted: Test group 3 (200 mg/kg bw/d):

Reduced mean food consumption (GD 6-8, GD 10-13 and GD 19-20; up to -10 % in comparison to the concurrent control) together with a decreased corrected body weight

gain (-15% compared to control) in combination with the following findings in pathology: thickened wall of the forestomach, thickened wall of the glandular stomach,

thickened wall of the duodenum, focus in the glandular stomach, deposition in the glandular stomach. No test substance-related adverse effects on gestational parameters or fetuses.

Test group 2 (50 mg/kg bw/d) and Test group 1 (15 mg/kg bw/d): No test substance-related adverse effects on dams, gestational parameters or fetuses

Therefore under the conditions of this prenatal developmental toxicity study, the oral administration of Sulfonium compounds, C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts) to

pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at a dose of 200 mg/kg bw/d caused signs of maternal toxicity. The combination of different individual findings (a reduction in food consumption, decrease in corrected body weight gain, thickened wall in stomach and duodenum) was assessed as adverse. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid dose of 50 mg/kg bw/d. The NOAEL for prenatal developmental toxicity is the highest tested dose of 200 mg/kg bw/d.