6-[(p-tosyl)amino]hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Remarks:

Source study for the purpose of read-across to the registered substance

Adequacy of study:

key study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 414. The study was conducted on 6-[(p-Tosyl)amino]hexanoic acid, which is the carboxylic acid component of the registered substance i.e. tosyl salt.

Justification for type of information:

The test substance was the carboxylic acid component of the registered substance (tosyl salt). Readacross between the tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered
substance is considered justified as the registered substance is manufactured directly from 6-[(pTosyl)amino]hexanoic acid by simple neutralisation with triethanolamine (TEA). Other than ionization of
the carboxylic acid group, the 6-[(p-Tosyl)amino]hexanoic acid remains chemically unchanged upon salt
formation. In water, the acid and amine components of 6-[(p-Tosyl)amino]hexanoic acid, compound with
2,2’,2’’-nitrilotriethanol (1:1) dissociate completely and behave essentially as independent substances.
Since TEA can be considered non-hazardous, it is the acid component of the salt that will have a
more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the
carboxylic acid group in 6-[(p-Tosyl)amino]hexanoic acid (pKa = 4.90) is the same in the free acid as
it is in the TEA salt. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the
same way whether it is in the salt form or as the parent carboxylic acid and hence it’s bioavailability
will be the same.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid

EC Number:

278-934-5

EC Name:

6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid

Cas Number:

78521-39-8

Molecular formula:

C13H19NO4S

Constituent 2

Reference substance name:

6-[(p-tosyl)amino] hexanoic acid

IUPAC Name:

6-[(p-tosyl)amino] hexanoic acid

Constituent 3

Reference substance name:

Tosyl carboxylic acid

IUPAC Name:

Tosyl carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

Full name of test item: 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid
Name in the study: ASCplus®
Lot Number: 1812004
CAS No.: 78521-39-8
EC No.: 278-934-5
Composition: 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid 74.76 %; water 25 %
Molecular formula: C13H19NO4S
Purity: > 99.5 % (6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid 74.76 %; water 25 %)
Appearance: White powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS:Nauki 6, Puschino, Moscow region, Russia 142290
- Age at study initiation: Approximately 12 weeks old at the initiation of dose administration on gestation day 5
- Weight at study initiation: 213 ± 11 g, N = 99
- Fasting period before study: Food consumption was assessed for each female quantitatively as g/kg of body weight/day by weighing of feeder (cage lid) at the beginning of the day and 24 hours after. Food consumption was recorded prior to the initiation of dose administration (gestation days 0-1), and at three-day intervals thereafter (gestation days 4-5, 7-8, 10-11, 13-14, 16-17 and 19-20).
- Housing: All animals were housed in solid bottom polycarbonate cages (Type IV, 598 х 380 х 200 mm (LxWxH), 2272 cm sq., Tecniplast s.p.a.) with bedding. Cages are equipped with steel lids, steel separators for the food and steel label holders. After identification and until mating, all females were housed by groups. Males were housed alone, and females cohabited with a male in the home cage of the male (2:1). After confirmed mating, dams were housed alone until euthanasia on gestation day 20 (G20).
- Diet : The animals were fed Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaten GmbH, Ferdinand-Gabriel-Weg 16, DE-59494 Soest, Germany) ad libitum.
- Water: Filtered tap water was provided ad libitum in standard water bottles.
- Acclimation period: During adaptation/acclimatization, animals were kept in groups (by litter/sex) in the barrier zone of facility and animal’s condition was evaluated daily by cageside observation. Before pre-mating oestrus cycle evaluation, all animals were identified by ear punch, weighed, and clinical observations were recorded. For all males, the testes were palpated, and for all females, the opened vagina was inspected visually. Animals considered unsuitable for the study were excluded prior to mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 °C to 24 °C
- Humidity (%): 30% to 70%
- Air changes (per hr): renewal of the room air at least 12 times hourly
- Photoperiod (hrs dark / hrs light): 08:00-20:00 - "Day", 20:00-08:00 - "Night

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dosage levels 100, 400, and 1600 mg/kg bw/day with 3-4-fold optimal interval were selected for the current study based on data for conducted screening toxicity study of the test item according OECD guideline 422.

The test item in the vehicle (water) was administered by gavage once daily to Sprague-Dawley female rats from day 5 to day 19 post-mating (inclusive). Three groups received the test item at dose-levels of 100, 400 or 1600 mg/kg bw/day. A concurrent vehicle control group received the vehicle (distilled water) on a comparable regiment and in the same volume of 10 mL/kg bw. Each group consists of 24-25 females with confirmed mating.

The vehicle and the test item were administered via an appropriately sized stainless steel ball-tipped dosing cannula connected with a syringe once daily. A separate cannula for each group was used. The dosage volume for all groups was 10 mL/kg body weight. Individual dosages were based on the last value body weights to provide the correct mg/kg bw/day dose.
During the dosing procedure, the formulation volume is continuously mixed on a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

Details on analytical verification of doses or concentrations:

The validated High-performance liquid chromatography (HPLC) method was used for the detection of the test item concentration in-vehicle formulations. The validation of the analytical method has been performed in the frame of 90-day oral toxicity study ofASCplus® (BTL BIBC study No. 704/20) where linearity, sensitivity (LLOQ), specificity/selectivity, accuracy, precision/repeatability, and formulation stability were assessed. Method validation report wias attached to the study No.704/20 final report.

The stability of the test item in the vehicle (water) prepared at concentrations of 10 and 160 mg/mL was confirmed following storage for 7 days at room temperature (the temperature range, 20 – 25 °C) during method validation study. Besides, homogeneity analysis was performed for formulations of 10 and 160 mg/mL after 4 days of storage after re-mixing. Results on formulation stability are presented in the report on analytical method validation (in the study 704/20 report).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: until evidence of mating by vaginal copulatory plug or the presence of sperm following a vaginal lavage.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

From day 5 to day 19 post-mating (days G5-G19)/ Three groups received the test item at dose-levels of 100, 400 or 1600 mg/kg bw/day.

Frequency of treatment:

Once daily

Duration of test:

From implantation to the day prior to scheduled caesarean section (day 5 to day 19 post-mating inclusive). On day 20 post-mating, the dams were sacrificed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 600 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3 groups of 24-25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels 100, 400, and 1600 mg/kg bw/day with 3-4-fold optimal interval were selected for the current study based on data for conducted screening toxicity study of the test item according OECD guideline 422.

All females were observed twice daily for mortality and morbidity and for signs of toxicity following dose administration. Body weights and food consumption are recorded at three-day intervals. On day 20 post-mating, the dams were sacrificed and subjected to a macroscopic examination and enumeration of corpora lutea. The weight of the thyroid gland, histopathological assessment of the thyroid gland, and assay of serum concentration of thyroxin (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH) were done in dams to observe pathological changes in thyroid function. Gravid uteri were weighed, and uteri content are examined to record implantation sites, early and late resorptions, dead, and live fetuses. The fetuses were weighed, sexed with measurement of anogenital distance (AGD), and submitted to external examination. Approximately half of the fetuses from each litter were subjected to a detailed examination of soft tissue by serial sectioning after fixation in Bouin’s solution while the other half underwent detailed skeletal examination following staining of bone with alizarin red and cartilage with alcian blue.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon at the same time, for morbidity and mortality. Each female was also observed for signs of toxicity approximately 30-45 minutes following dose administration. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual female body weights were recorded during animal identification, at day of confirmed mating and group assignment (gestation day 0), on the first day of dose administration (gestation day 5), and at three-day intervals thereafter (gestation days 8, 11, 14, 17, and 20 as the day of euthanasia). Body weight value on gestation day 20 was corrected for gravid uterine weight to calculate maternal body weight change

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was assessed for each female quantitatively as g/kg of body weight/day by weighing of feeder (cage lid) at the beginning of the day and 24 hours after. Food consumption was recorded prior to the initiation of dose administration (gestation days 0-1), and at three-day intervals thereafter (gestation days 4-5, 7-8, 10-11, 13-14, 16-17 and 19-20).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: A complete necropsy was conducted on all females at scheduled termination. Necropsy included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera.
Hysterectomy and examination of uterine content was done for all females. Ovaries were examined to determined number of corpora lutea. Gravid uterine weight was collected at scheduled necropsy. Uteri, which appear non-gravid by macroscopic examination, were opened and placed in 10 % ammonium sulfide solution for detection of early implantation loss. Thyroid glands were preserved and weighed after fixation.

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

Blood sampling:

Blood samples were collected from all females at the scheduled necropsies (as part
of euthanasia on day 20 of post-mating).
Animals were not fasted prior to blood collection. The blood was collected terminally
following anesthesia (Telazol® / Xyla®) from the caudal vena cava after laparotomy
using a syringe with 23G needle. Blood collection was done on the first part of the
day (within 10:00 – 13:00 hours) in randomized order to avoid bias.
The blood sample was placed in a tube without anticoagulant. The blood was
allowed to clot for 50 min and centrifuged (1600 x g, 4 °C, 15 min) for serum
separation. Serum from each animal was divided into 6 aliquots (for two aliquots for
each of T4, T3 and TSH analysis) and immediately frozen at –70 °C until assayed.

Fetal examinations:

- External examinations: Yes: all per litter
After cesarean section, all fetuses were subjected to external examination. Half of the fetuses from each litter was examined for skeletal abnormalities and the remaining for soft tissue alterations.
The fetal findings were described according to the harmonized terminology of the International Federation of Teratology Societies (IFTS) without categorization and classified as malformations or variations [Makris S.L. et al., 2009]:
malformation (major abnormality) refers to structural change considered detrimental to the animal (may also be lethal) and is usually rare; variation (minor abnormality) refers to structural change considered to have little or no detrimental effect on the animal; may be transient and may occur relatively frequently in the control population.
Findings of unknown significance for fetal development were also recorded, and their incidence data are presented and discussed in the study report.
The reproductive tract was examined with particular attention, and external sex was compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue malformations). In addition, male fetuses were examined for undescended testes.
External Examination, Body Weight and AGD:
Each live fetus was weighed and sexed with measurement of anogenital distance (AGD). All fetuses were subjected to a detailed external examination for gross anomalies with a recording of malformations and variations or non-classified findings. Fetuses were then anaesthetized by subcutaneous injection of Telazol® + Xyla® mixture and fixed in 96 % ethanol (approximately one-half of litter) for skeleton examination or Bouin’s fixative (remainder of litter) for soft tissues examination.
- Soft tissue examinations: Yes: half per litter
The remaining live fetuses in each litter were fixed with Bouin’s fluid. A detailed soft tissue examination was performed according to a free-hand serial sectioning technique, which will include the observation of all the organs and structures of the head, neck, thorax and abdomen.
Organs within the abdomen were examined in unsectioned abdominal region with particular attention on reproductive tract; an indication of incomplete testicular descent was noted in male fetuses.
- Skeletal examinations: Yes: half per litter
Approximately half of the live fetuses in each litter were fixed in ethyl alcohol, eviscerated and skinned for subsequent double staining of cartilage and bone with alcian blue and alizarin red.
A detailed examination of the skeleton included the observation of all the bone and cartilage structure of the head, spine, rib cage, pelvis, and limbs.
During evisceration, the reproductive tract was examined, and external sex was compared with internal (gonadal) sex; an indication of incomplete testicular descent was noted in male fetuses.
- Anogenital distance of all live rodent pups: AGD, mm, divided to the cube root of the body weight

Statistics:

All statistical tests were performed using Microsoft Excel (descriptive statistics) and statistical software Statistica for Window v.7.1 to compare the treated groups to the control group. The litter is accepted as an experimental unit for statistical analysis.

Continuous data variables (mean body weights and food consumption data) were analyzed by multi-factor analysis of variance ANOVA-2, followed by the Duncan test, to determine inter-group differences. Former implantation sites, number of corpora lutea, implantation loss indices, hormones concentration value, uterine, and thyroid weights were analyzed by parametric one-way analysis of variance (ANOVA). If the results of the ANOVA were significant (p<0.05), Dunnett's test is applied to the data to compare the treated groups to the control group. The t-test was used additionally to compare each dose group with the control value. Number of male and female fetuses per litter, AGD value, and mean value of affected fetuses per litter were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference and the t-test was applied to the AGD value to compare each dose from the control value.
The fetal body weight was analyzed by sex as well as for both sexes combined using a one-way analysis of variance (ANOVA) as described above and the t-test was applied to compare each dose from the control value. Additionally, statistical analysis for fetal body weight was done using analysis of covariant with litter size as a covariant.

Descriptive data, percentage values, and pathomorphological data were analyzed by Fisher's Exact Test; the percentage of pre-implantation loss was analyzed by Yates' corrected Chi-square test.

Indices:

Former implantation sites, number of corpora lutea, implantation loss

Historical control data:

Historical control data were used for:
-UTERINE CONTENT
-BODY WEIGHTS, ANOGENITAL DISTANCES AND SEX OF FETUSES
-FETUSES EXTERNAL OBSERVATIONS
-FETAL SOFT TISSUE EXAMINATION
-FETAL SKELETAL AND CARTILAGE EXAMINATION

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No test item related clinical findings were revealed in any dose groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Pregnant females treated with 400 and 1600 mg/kg bw/day doses had lower body weight gain due to the lower weight of the gravid uterus. The lower gravid uterine weight was correlated to the slight decrease in the mean number of fetuses per uterus and an increase in pre-implantation loss for both these doses.
400 mg/kg bw/day group: Gravid body weight gain: decrease on 5.7 % (p < 0.05). Gravid uterine weight: decreased on 10.4 % (p < 0.05)

1600 mg/kg bw/day group: Gravid body weight gain: decrease on 5.8 % (p < 0.05). Gravid uterine weight: decreased on 10.9 % (p < 0.05)

See attachment 'SUMMARY BODY WEIGHT AND BODY WEIGHT GAIN DATA FOR PREGNANT FEMALES'

Food consumption and compound intake (if feeding study):

no effects observed

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:

effects observed, treatment-related

Description (incidence and severity):

The absolute and relative thyroid weight of pregnant females was not significantly changed in
all dose test item treated group.
Histological alterations in the thyroid gland, which were considered to be associated with the
test item treatment were observed in the 1600 mg/kg bw/day dose group. In two females
from this group, the degeneration of follicular epithelium of minimum grade was found in
single follicles. C-cell hyperplasia of the slight grade was observed in one female.
Degeneration of follicular epithelium correlated to the slight decrease in thyroid hormones in
the high dose group. However, these findings were of minimum grade, found in single
females, and considered non-adverse.
The incidence of the ultimobranchial cyst was approximately the same among all groups,
including control. This finding is considered congenital, not treatment-related, as well as two
findings of ectopic lymphoid tissue in the low dose group

ASCplus lowered thyroid hormone levels in pregnant females. The decrease in T3 was dose-dependent and statistically significant compared to the control group in the 400 mg/kg
bw/day dose group (by 7.3 %, p < 0.05) and 1600 mg/kg bw/day dose group (by 7.9 %, p < 0.01). The decrease in T4 level was observed in the 1600 mg/kg bw/day dose group (by 5.6 %, p < 0.05).
Despite the increase in the mean value of TSH in the 1600 mg/kg bw/day dose group (by 23.5 % versus value in the control group), this change was not significant due to the high variation in the measurement hormone level. There were no indications of TSH-mediated thyroid gland activation, such as an increase in thyroids weight and histological changes.
Thus, the test item ASCplus lowered the thyroid hormones level in pregnant rat females in doses exceeding 400 mg/kg bw/day. The accompanying rise in TSH was slight and nonsignificant on 1600 mg/kg bw/day dose without apparent changes in thyroid glands.

See attachment 'Endocrine findings'

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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):

All treated females were sacrificed during a scheduled necropsy on post mating day 20. During necropsy, there were no gross findings related to the test item administration.
Females No.52 and No.92 (group 4) were without fetuses in the uterus. In female No.52, the four implantation sites with early resorptions were observed; however, both uteri were stained with 10% ammonium sulfide. There were no implantation sites in female No.92, and two additional sites were revealed in female No.52. Female No.41 (group 4) had only one fetus, and one other site of implantation was revealed after staining with ammonium sulfide.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological alterations in the thyroid gland, which were considered to be associated with the test item treatment were observed in the 1600 mg/kg bw/day dose group. In two females from this group, the degeneration of follicular epithelium of minimum grade was found in single follicles.

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The mean number of corpora lutea and implantation sites did not statistically differ in the test item treated groups compared to the control vehicle group. However, the mean number of implantation sites was slightly decreased by 8.6 % and 7.0 %, respectively, in the 400 and 1600 mg/kg bw/day dose groups.
The mean number of pre-implantation loss per females was not statistically changed in the test item treated groups; however, the percentage of pre-implantation loss was increased approximately equally in the 400 and 1600 mg/kg bw/day dose group (23.3 % and 21.5 % compared 12.1 % in the control vehicle group, p < 0.01). This increase was not so obvious when compared with the historical reference value 17.8 % of pre-implantation loss (136 implantations of 764 total corpora lutea, see Table S9-2 in Section 11.3.3), but was also statistically significant in the 400 mg/kg bw/day dose group (p < 0.05, Yates corrected Chisquare test).
According to previous studies, implantation sites in rats can be visualized (using intravenously Evans blue) 5 days 12 hours later mating [Novaro V. et al., 2002; Hamilton G. et al. 1994]. In our study, the test item administration started at this time (approximately 5.5 days after mating). In some females, this pre-administration period could be some longer (maximum 5 days 17 hours) or less (5 days 5 hours). Therefore, it is likely that the first
administered dose of 400 and 1600 mg/kg bw/day can adversely affect the implantation process.
The total post-implantation loss in the test item treated group did not significantly differ compared control vehicle group. The number of females with post-implantation loss was approximately the same in all groups. However, the mean percentage value was slightly increased in the 1600 mg/kg bw/day dose group (7.0 % versus 3.6 % in the control vehicle
group and 4.7 % as historical control value) and correlated to the slight increase in the percentage of early resorptions.
Thus, in the 400 mg/kg bw/day dose group, the slight decrease in the mean number of fetuses per animal is mainly associated with a change in implantation. In the 1600 mg/kg
bw/day dose group, the slight decrease in the mean number of fetuses per uterus is due to the changed implantation as well as a slight increase in post-implantation loss on the early stage.

See attachment 'SUMMARY DATA OF UTERINE CONTENT

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

The late resorptions were observed only in the test item treated groups with the maximal percentage value of 1.8 % in the 100 mg/kg bw/day dose group. However, this increase was not apparent comparing to the historical control value (1.3 %). Considering that a slight increase in the percentage of late resorptions was not dose-dependent, it is assumed to be a test item unrelated.
The percentage and the mean number of early resorptions were not statistically changed compared to the control vehicle group. However, in the 1600 mg/kg bw/day dose group, the mean values of early resorptions were notably increased compared to the other groups as well as the historical control data.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The test item did not cause morbidity and mortality of pregnant females as well as any clinical signs.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

pre and post implantation loss

other: endocrine findings (migrated information)

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

In the 100 and 400 mg/kg bw/day dose groups, the body weight of male and female fetuses as well as mean fetal body weight did not significantly differ from the values in the control vehicle group. In the 1600 mg/kg bw/day dose group, the body weight of female fetuses was reduced by 5.0 % compared to the control vehicle group (p < 0.05), and a tendency to decrease in the male body weight was observed (by 3.6 % ).
In the 1600 mg/kg bw/day group, fetal body weight: decreased (by 5.0 % for female, p < 0.05, and by 3.6 % for male, non-significantly)

See attachment 'SUMMARY RESULTS OF BODY WEIGHTS, ANOGENITAL DISTANCES AND SEX OF FETUSES'

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect on mean number and percent of live offspring.
See attachment 'summary data of uterine content'

Changes in sex ratio:

effects observed, treatment-related

Description (incidence and severity):

The mean number of males per litter was statistically reduced compared to the control vehicle group in the 400 and 1600 mg/kg bw/day dose groups (p < 0.05). The percentage of males per litter was decreased in the 400 mg/kg bw/day dose group (45.3 %) and the 1600 mg/kg bw/day dose group (44.4 %) compared to the value in the control group (50.7 %). This decrease in the ratio of male and female fetuses was not statistically significant but also
notable as compared to the historical control value (54.1 % of males, Table S13-2)
400 mg/kg bw/day group % males per litter: decreased (45.3 % versus 50.7 % in the control group, p < 0.05)

1600 mg/kg bw/day group % males per litter: decreased (44.4 % versus 50.7 % in the control group, p < 0.05)

See attachment '. SUMMARY RESULTS OF BODY WEIGHTS, ANOGENITAL DISTANCES AND SEX OF FETUSES'

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

effects observed, treatment-related

Description (incidence and severity):

The absolute and normalized anogenital distance in male fetuses was not significantly changed in all dose groups. In female fetuses, the normalized AGD value was slightly but significantly increased in the 1600 mg/kg bw/day dose group compared to the control vehicle and the mean historical control values (p < 0.05).
See attachment 'Body Weight, Sex, and Anogenital Distance of Fetuses'

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related external malformations were revealed in all dose groups. In the 100 mg/kg bw/day dose group, two fetuses from one litter had an umbilical hernia. Although there is no incidence of umbilical hernia in the historical control data, two fetuses of one litter from the control vehicle group also had an umbilical hernia. All umbilical hernia findings were associated with generalized subcutaneous edema (anasarca), which considered the finding
of “gray zone” with an unknown significance within the IFTS terminology. In total, anasarca was observed in 3 fetuses in the lower dose group (from one litter no.47) and four fetuses from the control vehicle group (from one litter no.23), and it is not considered to be
associated with the administered test item. One fetus from the 1600 mg/kg bw/day dose
group had a thread-like tail. Such malformation was single among all fetuses from the test item treated groups, and a similar tail defect (lack of tail) was found in one fetus from the control vehicle group. Other external observations were of unknown importance (“gray zone” of IFTS terminology), and were assumed without test item relation. The total affected fetuses were approximately the same among all groups. Single small fetuses, some of which were pale, were observed in all groups treated with the test item. Considering the historical control data with incidence of small fetuses and the absence of a direct relationship between this finding and the mean fetal body weight in the test item treated groups, it is not considered to be test item related.
In four fetuses from the 400 mg/kg bw/day dose group, thin skin prone to touch lesion was
noted. This finding of low incidence was not correlated to the individual fetal body weight, not
observed in the high dose treated group, and is assumed not to test item related.
Forelimb hyperflexion was observed in all dose treated groups with a slightly higher fetal
incidence in the 1600 mg/kg bw/day dose group. Simultaneously, in some fetuses in the low
BTL BIBC RAS Study Number: 705/20
Study Report ASCplus® , Prenatal Toxicity
Volume 1 of 2, Page 39 of 122
and high dose groups, but not in the control group, hindlimb hyperextension was noted.
Hyperflexion and hyperextension were observed with an equally high incidence in all groups,
including the control. Therefore, these findings of fore- and hindlimb hyperextension or
hyperflexion are not considered to be related to the test item, but rather are caused by the
fetus's functional state during cesarean section and as an assumption by anesthesia.
Additionally, a domed head was noted with approximately the same incidence in all groups,
did not correlate with findings in the skull and brain, and was probably associated with the
fetal muscle tone (the effect of the lowered head). The historical control dalso has the incidence of the fetal domed head, which also did not correlate to the skeletal and visceral findings. The bent tail was also noted in historical control data. Although the incidence of a bent tail in the study was higher than the historical control value, it was approximately the same in all groups and was not associated with any skeletal findings.
There was no correlation among the slight increase in AGD in female fetuses in the high dose group and external observation of genital tubercle. Fetal and litter incidence of large genital tubercle in females did not significantly change in the 1600 mg/kg bw/day dose group.

See attachment RESULTS OF FETUSES EXTERNAL MALFORMATIONS DATA

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Exposure to doses of ASCplus of 400 or 1600 mg/kg bw/day was associated with reduced ossification in the fetuses; furthermore, the split sternum was observed in one fetus in the 1600 mg/kg bw/day dose group.
In the 400 mg/kg bw/day dose group, the slight increase in fetal incidence of incomplete ossification in interparietal skull bone (21.0 % versus 12.1 %, p < 0.05) and fifth metacarpal (47.1 % versus 36.2 %, p < 0.05) was observed. The incidence for total fetuses with incomplete ossified skull bones was increased compared control group (22.5 % versus 14.1 %, p < 0.05).
In the 1600 mg/kg bw/day dose group, the increase in fetal incidence of incomplete ossified frontal (17.2 % versus 9.4 %, p < 0.05), interparietal (20.9 % versus 12.1 %, p < 0.05), fifith metacarpal (48.5 % versus 36.2, p < 0.05), and total unossified phalanges of forepaws (56.0 % versus 39.6 %) was noted. The incidence of total affected fetuses in skull and forepaw was increased to 23.9 % (p < 0.05) and 82.8 % (p < 0.01), respectively, compared to 14.1 % and 66.4 % values in the control vehicle group.
Sternoschisis (split sternum) founded in one fetus in the high dose group was not previously observed in the historical control population. Therefore, despite the uniqueness of the finding, its relation to the test item cannot be excluded.
One fetus in the control vehicle group (No.77-14f) recorded without tail during external observation, had only one unaltered sacral vertebrae and two caudal vertebrae. Excluding discussed malformations, for test item related findings of altered ossification, cartilages were present, suggesting that these skeletal variations were due to delayed ossification rather than to a persistent alteration. In the 1600 mg/kg bw/day dose group, the delayed ossification correlated to the decrease in fetal body weight (significant in females
and non-significant for males, Section 11.5.1). However, reduced ossification of the skull and 5th metacarpal was observed among the fetuses exposed to 400 mg/kg bw/day dose, for which body weight was in the normal range. In this context, it is interesting that exposure to 400 or 1600 mg/kg bw/day dose reduced serum triiodothyronine and thyroxine levels. It is likely that the disturbances in maternal thyroid hormone homeostasis contribute to the
reduction in fetal skeletal ossification that was observed. The delayed appearance of ossification centers is a frequent finding in newborns with congenital hypothyroidism, and reduced radiological ossification centers were found in the fetuses of dams thyroidectomized
prior to mating [Gil-Garay et al., 1991].
Thus, the test item ASCplus at the doses starting from 400 mg/kg bw/day reduces ossification in the fetuses, which correlated to the disturbances in maternal thyroid hormone homeostasis.
400 mg/Kg bw/day group Skeletal variations – Forelimb: increase in fetal incidence of incomplete ossification in 5th metacarpal (p < 0.05)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in interparietal bone (p < 0.05)

1600 mg/kg bw/dayn group Skeletal malformation: Sternum – split (one fetus)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in frontal bone (p < 0.05)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in interparietal bone (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of incomplete ossification in 5th metacarpal (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of unossified phalanges (p < 0.01)

See attachment DATA OF FETAL SKELETAL MALFORMATIONS

Visceral malformations:

no effects observed

Description (incidence and severity):

No test item related malformations of soft tissues were found in any treatment fetuses. One fetus from 1600 mg/kg bw/day dose group was without one eye. Despite the absence of this finding in historical control data, it is known that the unilateral absence of the eye can occur in this rat population. In the control group, the gastroschisis was observed in one fetus (No.23-5f). This fetus was small, had generalized edema and “gray zone” abnormalities in some visceral organs. Another fetus with generalized edema from this control litter (No. 23-1m) was examined for skeletal abnormalities. The fetal findings in the control group are regarded as random events. In the 100 mg/kg bw/day dose group, three fetuses from one litter (No.47) had generalized edema with an umbilical hernia in two of them. No malformations in soft tissues were revealed in fetuses of this litter. The test item did not cause an increase in the incidence of other findings in fetal soft tissues.
Most of these findings were alterations of “gray zone” with unknown significance. The fetal and litter incidence was approximately the same among all groups, including control, so they are not considered treatment-related.

See attachment DATA OF FETAL SOFT TISSUE MALFORMATIONS

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

skeletal malformations

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull sutures

skeletal: forelimb

Description (incidence and severity):

The test item at the doses starting from 400 mg/kg bw/day reduces ossification in the fetuses, which correlated to the disturbances in maternal thyroid hormone homeostasis.
400 mg/kg bw/day group: Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in interparietal bone (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of incomplete ossification in 5th metacarpal (p < 0.05)

1600 mg/kg bw/day group: Skeletal malformation: Sternum – split (one fetus)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in frontal bone (p < 0.05)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in interparietal bone (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of incomplete ossification in 5th metacarpal (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of unossified phalanges (p < 0.01)

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

400 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Daily dosages of 100, 400, and 1600 mg/kg bw of the test substance were administered by gavage once daily to three groups of Sprague-Dawley female rats from day 5 to day 19 post-mating (inclusive). A concurrent vehicle control group received the vehicle (distilled water) on a comparable regiment and in the same volume of 10 mL/kg bw. Each group consists of 24-25 females with confirmed mating.

The test item did not cause morbidity and mortality of pregnant females as well as any clinical signs. Under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity and embryo-fetal development of 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid (ASCplus®, CAS 78521-39-8) is considered to be 100 mg/kg bw/day.

Executive summary:

The potentially toxic effects of the test item 6-[[(4methylphenyl)sulphonyl]amino]hexanoic acid (ASCplus®, CAS 78521-39-8) on pregnancy and embryo-fetal development in rats was evaluated by an OECD TG 414. Daily dosages of 100, 400, and 1600 mg/kg bw of the test substance were administered by gavage once daily to three groups of Sprague-Dawley female rats from day 5 to day 19 post-mating (inclusive). A concurrent vehicle control group received the vehicle (distilled water) on a comparable regiment and in the same volume of 10 mL/kg bw. Each group consists of 24-25 females with confirmed mating.

The test item did not cause morbidity and mortality of pregnant females as well as any clinical signs. Pregnant females treated with 400 and 1600 mg/kg bw/day doses had lower body weight gain due to the lower weight of the gravid uterus. The lower gravid uterine weight was correlated to the slight decrease in the mean number of fetuses per uterus and an increase in pre-implantation loss for both these doses. The slight increase in pre-implantation loss is supposed to be due to the adverse effect of the first dose of ASCplus® administered on gestation day 5 during the implantation window in some females. Moreover, in the 1600 mg/kg bw/day dose group, the slight decrease in the mean number of fetuses was also correlated to the slight increase in post-implantation loss on the early stage.
In the 100 mg/kg bw/day dose group, there were no fetal alterations.

In the 400 mg/kg bw/day dose group, with normal fetal body weight, the ossification of some bones was delayed; the percentage of males per litter was decreased.
In the 1600 mg/kg bw/day dose group, delayed ossification was associated with reduced fetal body weight and decreased percentage of males per litter.
Delayed ossification in fetuses correlated to the disturbance in maternal homeostasis of thyroid hormones.

Therefore, under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity and embryo-fetal development of 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid (ASCplus®, CAS 78521-39-8) considered to be 100 mg/kg bw/day.
A summary of the dose descriptors of maternal and embryo-fetal toxicity is attached.

The test substance was the carboxylic acid component of the registered substance (tosyl salt). Read-across between the tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered substance is considered justified as the registered substance is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid by simple neutralisation with triethanolamine (TEA). Other than ionization of the carboxylic acid group, the 6-[(p-Tosyl)amino]hexanoic acid remains chemically unchanged upon salt formation. In water, the acid and amine components of 6-[(p-Tosyl)amino]hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) dissociate completely and behave essentially as independent substances. Since TEA can be considered non-hazardous, it is the acid component of the salt that will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the carboxylic acid group in 6-[(p-Tosyl)amino]hexanoic acid (pKa = 4.90) is the same in the free acid as it is in the TEA salt. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the same way whether it is in the salt form or as the parent carboxylic acid and hence it’s bioavailability will be the same.