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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Jul 2020 - 17 Aug 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2018
Deviations:
yes
Remarks:
criteria used for categorising fetal external, soft tissue and skeletal examination not specified, only a limit set of historical control data provided, short acclimatisation period as time-mated females were purchased for the study
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
EC Number:
263-174-9
EC Name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
Cas Number:
61791-42-2
Molecular formula:
UVCB
IUPAC Name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts

Test animals

Species:
rat
Strain:
other: Crl:WI(Han
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, United Kingdom
- Age at the time of mating: 9 – 11 weeks
- Weight at dosing: 180.6 – 258.2 g
- Housing: Individually in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes. Aspen wood chips were used as bedding.
- Diet: VRF1 diet (Special Diets Services Ltd., Witham, United Kingdom), ad libitum
- Water: Main tap supply water, ad libitum
- Acclimation period: The animals were delivered to the testing laboratory by gestation Day (GD) 3. Acclimatisation was limited by mated status, but all animals were given a clinical inspection for ill health upon arrival and before the start of dosing to ensure suitability for the study.

DETAILS OF FOOD AND WATER QUALITY:
Each batch of diet and the water was periodically analysed for specific constituents and contaminants. No contaminants were present in the diet and in the water at levels that might have interfered with achieving the objective of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Jul 2020 To: 17 Aug 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was formulated as a solution in purified water. The test article was slowly added to purified water whilst stirring for approximately 10 minutes. Formulations were prepared weekly and stored at room temperature (15 - 25°C) in a sealed container.
VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test item in the vehicle were confirmed in a previous study and found to be stable over a period of 14 days for formulations of 1 and 250 mg/mL at 15 - 25°C (Covance study no. 8422999). In the present study, formulations prepared for use at the beginning (gestation Day (GD) 6 and end of dosing (GD 20) were analysed to determine the achieved concentration. Triplicate samples were taken from the middle of the test article formulations and were analysed.
The mean achieved concentrations were 110% (GD 6) and 100 - 104% (GD 20) of the nominal concentration with relative standard deviations ranging from 0.33 – 0.53% (GD 6) and 0.28 – 0.60% (GD 20). The values fell into the accepted range of 90 – 110% of the nominal concentration with a relative standard deviation of ≤ 5.0% and were therefore considered acceptable.
Details on mating procedure:
The females were mated prior to arrival in the testing facility.
Duration of treatment / exposure:
Days 6 - 21 of gestation.
Frequency of treatment:
daily, 7 days/week
Duration of test:
Day 21 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for the main developmental toxicity study were selected based on the results of a preliminary dose-finding study (Covance study no. 8417090). Pregnant rats were administered dose levels of 100, 300 and 1000 mg/kg bw/day. The test article was well tolerated at all dose levels, with only transiently lower food consumption observed following administration of 1000 mg/kg bw/day during early pregnancy. No fetal variations or malformations were noted; as such, the limit dose of 1000 mg/kg bw/day was considered suitable as the high-dose level for use in the present study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily (at the beginning and end of each working day)
- Cage side observations included: mortality, clinical signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination on gestation Days (GD) 3, 6, 7, 8, 9, 12, 15, 17, 19, 20, and 21.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 6, 7, 8, 9, 12, 15, 17,19, 20, and 21. An additional, unscheduled body weight was recorded on GD 20 for 1/20 females at 100 mg/kg bw/day.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The amount of food consumed was determined from GD 6 - 7, 7 - 8, 8 - 9, 9 - 12, 12 - 15, 15 - 17, 17 - 19, 19 - 20, and 20 – 21.

WATER CONSUMPTION: No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 21
- Organs examined: Ovaries and uteri

OTHER:
Thyroid Hormone analysis:
- Time schedule for collection of blood: Blood samples were collected from the abdominal aorta at scheduled necropsy on gestation Day 21.
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: all animals in all groups
- Parameters examined: triiodothyronine (T3), thyroxin (T4) and thyroid stimulating hormone (TSH)

Thyroid weights were recorded from the parental females of all groups at scheduled caesarian section.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Pregnancy status, terminal body weight and number of dead fetuses.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
- Other: Anogenital distance and sex ratio
Statistics:
For details on statistical analysis please refer to the document "Statistical analysis_OECD 414" under "Attached background material".
Indices:
Maternal parameters:
Pre-implantation loss (%) = ((Number of corpora lutea – Number of implantations)/Number of corpora lutea) x 100
Post-implantation loss (%) = ((Number of implantations – Number of live embryos)/Number of implantations) x 100
Corrected body weight (Carcasse weight) = Terminal body weight – uterine weight
Corrected weight change = Carcasse weight – gestation Day 6 body weight
Total weight change = Gestation Day 21 body weight – gestation Day 6 body weight

Litter data:
Male fetuses (%) = (Number of male fetuses/Number of fetuses of determined sex) x 100
Historical control data:
Historical data was provided for fetal skeletal variations only to allow comparison with concurrent controls.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Excessive salivation was noted in the animals at 100 mg/kg bw/day and above (in 4/20, 6/20 and 10/20 at 100, 300 and 1000 mg/kg bw/day). In addition, mouth rubbing was observed in animals of the mid (7/20) and high dose groups (20/20). The observations were made immediately after dosing, but were also observed in some animals at the end of the day observations of gestation Day (GD) 20. Isolated incidences of paddling behaviour was noted for 1/20 and 2/20 animals on GD 13 or 15 at 300 and 1000 mg/kg bw/day. The findings were attributed to unpalatability or the unpleasant taste of the test article and therefore considered non-adverse. For details please refer to Table 1 under “Any other information on results incl. tables”.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was a statistically significant reduction in body weight gain observed for gestation Days (GD) 9 – 12, 20 – 21 and over the entire treatment period (GD 6 – 21). Initial body weight losses were also observed for the animals of this group, but did not reach statistical significance. The findings were consistent with a reduced food consumption in the animals of this group and attributed to treatment. However, in absence of a detrimental effect on pregnancy or the developing fetus, these findings were considered non-adverse.
No effect on body weight or body weight change was noted following administration of 100 or 300 mg/kg bw/day. For details please refer to Table 2 under “Any other information on results incl. tables”.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day there was a statistically significant reduction in food consumption observed from the start of dosing up to gestation Day (GD) 15 and on the last day of dosing (GD 21). Overall mean food consumption was -15% when compared to control animals. The findings were in line with a reduced body weight development in these animals and related to treatment. However, in the absence of a detrimental effect on pregnancy or the developing fetus, the findings were considered not to represent an adverse effect of the test article.
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
Endocrine findings:
not specified
Description (incidence and severity):
The following ED-related parameters were investigated in the study: T4 and TSH level, anogenital distance, genital abnormalities, thyroid weight and histopathology, gravid uterus weight, foetal development, litter size, litter/pup weight, number of implantations/ corpora lutea, number of embryonic or foetal deaths and viable foetuses, post-implantation losses, pre-implantation losses, presence of foetal anomalies (external, visceral and skeletal) and sex ratio.
For details, please refer to the respective result fields and the endpoint summary.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test article-related macroscopic findings were recorded for the thyroid of animals administered 100, 300, or 1000 mg/kg bw/day. All tissues were macroscopically unremarkable, or the findings observed were generally consistent with the usual pattern of findings in rat of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test article-related microscopic findings were recorded for the thyroid of animals administered 100, 300, or 1000 mg/kg bw/day. All tissues were microscopically unremarkable, or the findings observed were generally consistent with the usual pattern of findings in rat of this strain and age.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyriod hormone levels were not affected by treatment.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
A total in utero litter loss was noted for 1/20 females at 100 mg/kg bw/day and for 1/20 females at 1000 mg/kg bw/day. The observations were within the laboratories historical control range, occurred without any dose response relationship and, in the absence of any pre- or post-implantation losses, was considered unrelated to treatment.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
1/20 females administered 100 mg/kg bw/day and 1/20 females administered 1000 mg/kg bw/day started parturition prior to the terminal sacrifice. An increased incidence of early parturition was recently observed in this strain of animal from the animal supplier; therefore, this finding was unrelated to the test article.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
1/20 females of the control group and 1/20 females administered 100 mg/kg bw/day were not pregnant. These animals were supplied non-pregnant, and, as such, the lack of pregnancy for these animals was unrelated to the test article.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were observed in 2 fetuses of the control group and in 1 fetus of the 1000 mg/kg bw/day group. Abnormal findings in the control group comprised a shortened 13th rib on the right in a female fetus and a male fetus showing a malrotated hindlimb on the right ankle joint, a malpositioned talus of the right hindlimb and hyperextention of the hindlimb on the right ankle joint. At 1000 mg/kg bw/day, a malrotated hindlimb of the ankle joint was observed bilaterally in 1 male fetus. The findings were considered to be incidental.
At 1000 mg/kg bw/day, reduced incidences of incomplete interparietal ossification of the skull was noted in the fetuses (1.93% of the fetuses) when compared to control animals (16.4% of the fetuses). In addition, incomplete metatarsal ossification of the hindlimbs was occasionally observed in animals of the control and test item-treated groups. The findings were within the range of the laboratories historical control data and thus considered to have arisen incidentally. For details please refer to Table 4 under “Any other information on results incl. tables”.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg bw/day, diaphragmatic hernia in the muscular region, protruding from the liver was noted in 1 male fetus. At 1000 mg/kg bw/day, 1 male fetus showed edema. The latter finding was considered a congenital abnormality. The fetus was also recorded with adrenal variations, abnormal blood vessels, and dilated brain ventricles, which was associated with the severe edema. The weight of this fetus was also greater than other fetuses maternally exposed at this dose (7.28 g), and the higher fetal weight was most likely due to increased fluid retention. This is an unusual finding; however, as this was a single finding, it was considered congenital and unrelated to the test article.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Summary of clinical signs of toxicity

Dose group (mg/kg bw/day) 0 100 300 1000
Mouth rubbing 0/20 0/20 7/20 20/20
Paddling 0/20 0/20 1/20 2/20
Excessive salivation, mild, clear 0/20 1/20 3/20 4/20
Excessive salivation, mild, colorless 0/20 0/20 3/20 4/20
Excessive salivation, moderate, clear 0/20 0/20 0/20 1/20
Excessive salivation, moderate, colorless 0/20 1/20 0/20 1/20

Table 2: Body weight and body weight gain

Dose group (mg/kg bw/day) 0 100 300 1000
Body weight (g)
BW GD 6 214.5 ± 19.66 211.4 ± 14.10 211.7 ± 14.17 211.4 ± 10.84
% control 100 99 99 99
BW GD 12 235.0 ± 23.15 234.8 ± 15.16 234.3 ± 14.23 226.8 ± 11.08
% control 100 100 100 97
BW GD 21 314.8 ± 32.94 313.0 ± 25.63 312.0 ± 23.41 295.9 ± 19.63
% control 100 99 99 94
Body weight gain (g)
GD 6-7 2.9 ± 4.27 2.9 ± 3.39 2.7 ± 4.49 1.4 ± 3.68
% control 100 100 93 48
GD 9-12 14.4 ± 4.15 16.0 ± 2.93 13.9 ± 4.30 10.6 ± 3.42**
% control 100 111 97 74
GD 17-19 22.9 ± 5.32 22.8 ± 6.00 21.8 ± 5.34 19.6 ± 6.74
% control 100 100 95 86
GD 20-21 15.8 ± 4.42 14.3 ± 4.20 15.0 ± 4.47 7.0 ± 11.93**
% control 100 91 95 44
GD 6-21 100.3 ± 16.89 101.2 ± 14.57 100.3 ± 19.81 83.5 ± 12.74**
% control 100 101 100 83
**: statistically significant at p < 0.01; BW: body weight; GD: gestation day

Table 3: Food consumption

Dose group (mg/kg bw/day) 0 100 300 1000
GD 6-7 20.3 ± 3.65 19.2 ± 3.15 19.4 ± 3.17 15.6 ± 3.14***
% control 100 94 96 77
GD 9-12 19.1 ± 2.60 19.9 ± 2.23 19.4 ± 2.41 14.9 ± 2.13***
% control 100 104 102 78
GD 15-17 21.5 ± 3.24 22.3 ± 2.39 22.2 ± 2.51 20.1 ± 2.12
% control 100 104 103 79
GD 20-21 20.6 ± 3.94 19.2 ± 3.17 18.8 ± 3.31 14.2 ± 5.98***
% control 100 93 91 69
GD 6-21 20.8 ± 2.33 21.0 ± 1.91 20.6 ± 1.78 17.6 ± 1.63***
% control 100 101 99 85
*, ** and ***: statistically significant at p < 0.05, p < 0.01 and p < 0.001; GD: gestation day

Table 4: Skeletal malformations in fetuses

Dose group (mg/kg bw/day) 0 100 300 1000 HCD
Skull
Interparietal: incomplete ossification
 Litter (%) 47 39 47 11 5 - 75
Fetal (%) 16.4 12.22 13.73 1.93** 0.76 - 49.17
Hindlimb
metatarsal: incomplete ossification Litter (%) 21 0 11 26 0 - 43
Fetal (%) 5.44 0 1.93 10.33 0 - 14.74
**: statistically significant at p< 0.01; HCD: historical control data generated in the testing facility (time frame for data generation not specified)

Applicant's summary and conclusion

Conclusions:
The test item had no effect on intrauterine development. The NOAEL for maternal and developmental toxicity were both 1000 mg/kg bw/day.