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Toxicological information

Developmental toxicity / teratogenicity

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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

Constituent 1
Chemical structure
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 : ad libitum
- Water: ad libitum
- 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 / concentrationsopen allclose all
Dose / conc.:
1 600 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
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
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- 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
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
400 mg/kg bw/day
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
Gravid body weight gain: decrease on 5.8 % (p < 0.05)
Gravid uterine weight: decreased on 10.9 % (p < 0.05)
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Behaviour (functional findings):
no effects observed
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
400 mg/kg bw/day
Implantation: reduced number of implantation sites (by 8.6 % non-significantly); increase in percentage of pre-implantation loss (by 23.3 %, p <0.01)

1600 mg/kg bw/day
Implantation: reduced number of implantation sites (by 7.0 % non-significantly); increase in percentage of pre-implantation loss (by 21.5 %, p <0.01)
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
1600 mg/kg bw/day
Implantation loss: increase in early resorptions (6.6 % versus 3.6 % in the control group, non-significant)

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
pre and post implantation loss
other: endocrine findings (migrated information)

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
1600 mg/kg bw/day
Fetal body weight: decreased (by 5.0 % for female, p < 0.05, and by 3.6 % for male, non-significantly)
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
400 mg/kg bw/day
% males per litter: decreased (45.3 % versus 50.7 % in the control group, p < 0.05)

1600 mg/kg bw/day
% males per litter: decreased (44.4 % versus 50.7 % in the control group, p < 0.05)
Changes in postnatal survival:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
400 mg/Kg bw/day
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/day
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)

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
fetal/pup body weight changes
skeletal malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull sutures
skeletal: forelimb
Description (incidence and severity):
400 mg/kg bw/day
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
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 (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes

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.


 

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