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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

The following information is taken into account for any hazard / risk assessment:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male and female rats (Zeljenkova, D (2013)). Treatment of males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period), treatment of females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period) for 54 days.

Additional satellite groups of 5 male and 5 female were treated with 0 and 1600 mg ASC plus/kg bw. and used for observation of reversibility, persistence or delayed occurrence of systemic toxic effects for at least 14 days post treatment.

The test-article was formulated in drinking water and administered in 10 ml/kg bw.

At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOAEL).

1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.

Zeljenkova, D (2013):

overall NOAEL: 400 mg/kg bw.

NOAEL for fertility effects: 1600 mg/kg bw.

 

OECD TG 414 Prenatal Developmental Toxicity Study:

Daily dosages of  0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of   24-25 female Sprague-Dawley rats  from day 5 to day 19 post-mating (inclusive). ASCplus® was formulated in 10 ml/kg bw. destilled water. (Oksana N. Khokhlova (2021) BTL BIBC RAS)

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 a slight decrease in the mean number of fetuses per uterus and an increase in pre-implantation loss for both these doses.

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.

overall NOAEL: 100 mg/kg bw.

NOAEL for fertility effects: 100 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in accordance to OECD guideline

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany, D-97633 Sulzfeld
- Age at purchase: 11 weeks
- weight rage at time of grouping: males, 175-200 g
- Fasting period before study: no
- Housing: 2 per cage,
- Cages: TECHNIPLAST filter top cages type 2145 F with an G-Temp (PSU) durable filter cover, 480x265x210 mm³, floor area 940 cm²,
- Source: Techniplast Company, Italy
- Diet: ad libitum, M3, BONAGRO Ltd., reg. CZ 10174, Czech Republic
- Water: ad libitum, tap water
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE: suspension in water
- Amount of vehicle: 10 ml/kg at similar times each day

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1 (probably) (male animals: see Endpoint study record in section 7.5.1 "Repeated dose Toxicity")
- Length of cohabitation: until copulation/ up to 14 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- M/F ratio per cage: 1/1
- Length of cohabitation: Each morning the females were examined for the presence of sperm in vaginal lavages
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually.
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period)
females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period)

Frequency of treatment:

daily by stomach tube

Details on study schedule:

- Age at mating of the mated animals in the study: 14 weeks

Remarks:

Doses / Concentrations:
actual ingested doses
Basis:
actual ingested
0, 100, 400, 1600 mg/kg bw./day

No. of animals per sex per dose:

12

Control animals:

yes

Details on study design:

- Control groups: drinking water
- Dose selection rationale: Results of an acute toxicity study with oral administration to male and female rats
- Result: no effects up to and including 2000 mg/kg bw.
- Rationale for animal assignment: randomly grouped
- Section schedule rationale: all animals were sacrificed

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on administration days 1, 8, 15, 22, 29, 36, 42, 50 and on day of autopsy, pups within 24 hours of parturition and on day 4 post-partum

FOOD CONSUMPTION : 2 weeks before mating, after 2 week mating period and weekly until the end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14 (before mating) and prior to autopsy from the satelite groups and from the control and high dose group.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:day 14 (before mating) and prior to autopsy from the satelite groups and from the control and high dose group.

URINALYSIS: No

Sperm parameters (parental animals):

Parameters examined in male parental generations: testis weight, epididymis weight, histopathology of testis and epidymidis

Litter observations:

PARAMETERS EXAMINED
numbers of pups born, delivery index, number of pups alive, birth index, live birth index, pup weight on day 0 of lactation, sex ratio, number of pups alive on day 4 of lactation, pup weight on day 4 of lactation, general status of pups, observation of external deformities, corpora lutea, implantations, visible resorptions, early resorptions


GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

Postmortem examinations (parental animals):

POST-MORTEM EXAMINATIONS: Yes

SACRIFICE
- Male animals after 54 days: All surviving animals
- Female animals: Sacrifice on lactation day 4 (starting with day 0),

ORGAN WEIGHTS
- Organ weights: brain, heart, thymus, spleen, liver, testis, epididymis, kidney, adrenal gland

HISTOPATHOLOGY: Yes: high dose and control animals
- Organ: medulla oblongata, brain, heart, pancreas + lymphnode, spleen, liver, lung, small intestine, stomach, kidneys, adrenal gland, testes, prostrate, urinary bladder, bone + bone marrow, thymus, trachea, white + brown fat, muscle, pituitary gland

Postmortem examinations (offspring):

Gross pathology: observation of external deformities

Statistics:

Statistical evaluation was operated using the software SPPS version 16.0. Group data were represented by mean, standard deviation and median. Statistical analysis in case of data measured once during the study (organ weight, haematology, clinical chemistry) : Mann-Whitney U test for pairwise comparison between control and individual experimental groups on significance level alpha = 0.05.
Statistical analysis in case of repeated data measurement (body weight, food intake): Repeated measures ANOVA (procedure General Linear Model (GLM) for Repeated Measures in SPSS statistical software).

Reproductive indices:

number of mated pairs, number of copulated pairs, copulation index, number of pregnant animals, fertility index, pairing days until copulation, frequency of vaginal estrus, number of pregnant females, number of pregnant females with pups alive, gestation index, gestation lenth in days, number of corpora lutea, number of imlantation sites, implantation index, number of pups born, delivery index

Offspring viability indices:

number of pups alive, birth index, live birth index, pup weight on day 0 of lactation, sex ratio, number of pups alive on day 4 of lactation, viability index, pup weight on day 4 of lactation, general status of pups, observation of external deformities

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS:
- 100, 400 and 1600 mg/kg bw/day:
no differences to the control animals observable

MORTALITY: no

BODY WEIGHT GAIN: 1600 mg/kg bw: reduced

FOOD CONSUMPTION: no statistical differences

HAEMATOLOGY/ CLINICAL CHEMISTRY:
Haematology and clinical chemistry reveales some statistically significant differences, but these were not related to dosage or not confirmed by the findings in other groups, for example by the results of the satellite groups, or the effects are of biological low relevance i.e..

URINALYSIS: not examined

NEUROBEHAVIOUR: not examined

ORGAN WEIGHTS: no statistical differences

GROSS PATHOLOGY: no dosage related effects

HISTOPATHOLOGY: NON-NEOPLASTIC: no statistical differences

HISTOPATHOLOGY: NEOPLASTIC: no statistical differences

HISTORICAL CONTROL DATA: not given

Dose descriptor:

NOEL

Remarks:

fertility

Effect level:

>= 1 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: P and F1 (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

CLINICAL SIGNS (OFFSPRING): no abnormalities in the general status

BODY WEIGHT (OFFSPRING): no significant differences between the control group and the various dosage groups werde observed in males or females in body weights on day 0

On lactation day 4 a higher number of dead pups was counted in the high-dose level group. Moreover, there was a stastitically significant but very low difference in the body weight between the control group and the 400 and the 1600 mg/kg bw dose groups. However, this difference is considered to be of low biological relevance.

GROSS PATHOLOGY (OFFSPRING): no external deformities were observed in the surviving pups on the day of birth. In autopsies of pups on lactation day 4, no abnormalities were observed in the control, nor were any abnormalities in the autopsy of the dead pup.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 other: mg/kg bw/day mother

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 600 other: mg/kg bw/day mother

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

gross pathology

Reproductive effects observed:

not specified

Conclusions:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male and female rats. Treatment of males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period), treatment of females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period) for 54 days.
Additional satelite groups of 5 male and 5 female were treated with 0 and 1600 mg ASC plus/kg bw. and used for observation of reversibility, persistence or delayed occurence of systemic toxic effects for at least 14 days post treatment.
The test-article was formulated in drinking water and administered in 10 ml/kg bw..
At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOAEL).
1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.
NOAEL: 400 mg/kg bw. However NOAEL (Fertility) = 1600 mg/kg bw.

Executive summary:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male rats for 54 days. The test-article was formulated in drinking water and administered in 10 ml/kg bw..

Histopathology and gross necropsy did not reveal any test article related changes.

The organ weights showed some statistically significant differences, but these were not related to dosage or not affirmed by the findings in other groups, for example by the results of the satellite groups.

The same is true for the statistical significant differences of the haematological parameters and the result of the clinical chemistry, especially decreased activities of the liver enzymes in the blood. These are merely statistical effects without biological relevance.

Two effects with a possible relation to the oral administration of ASC plus were observed:

- At birth no differences were observed between the groups, but on day 4 after birth a higher number of dead pups was counted in the high-dose level group.

- With male rats a dose dependent decrease in body weight is observed. This effect is in the highest dose group statistically significant and confirmed by the satelite group but not confirmed by the body weight development of the females and not confirmed by the food consumption of males and females.

Although an influence of an infection with paracites seems possible and may explain both effects, a non-specific influence of ASC plus cannot be excluded. The daily administration of a suspension of ASC plus in 10 ml/ kg bw. may have led to a reduced appetite of the animals. This should be especially true for the highest dosage group, as here the suspension was relatively dense. Imaginable is also an osmotic effect of non-resorbed ASC plus in the intestine. At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOEL).

1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.

With regard to effects on fertility of the present study 1600 mg ASC plus/kg bw. per day can be defined as "No Observed Adverse Effect Level" (NOAEL).

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable guideline study performed under GLP.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

According to the present data the toxicity and the toxicokinetics of ASC plus (6-[[(4-methylphenyl) sulphonyl]amino]hexanoic acid) can be described as follows:

1. At dosages of 100 mg/kg bw. the animals showed no differences to the control animals (NOAEL) (OECD 414).


2. 400 mg ASC plus/kg bw. (OECD 408) may have induced:

with Males: increased serum urea and Leydig cells hyperplasia in the testes,

With Females: decreased APTT, increased liver weights; liver, fatty change; kidney, lipofuscinosis, calculi in pelvis; thyroid glands, C-cell hyperplasia.

3. 1600 mg ASC plus/kg bw. (OECD 408) may have induced:

Males, 1600 mg/kg bw/day: increased urobilinogen, decreased urine pH, decreased serum triglyceride, increased serum urea, increased liver weights and kidney weights; liver, fatty change; kidney, lipofuscinosis; thyroid glands, C-cell hyperplasia; testes, Leydig cells hyperplasia;

Females, 1600 mg/kg bw/day: increased urobilinogen, decreased: APTT, increased liver weights and kidney weights; liver, fatty change, hepatocellular hypertrophy; thyroid glands, C-cell hyperplasia.

4. 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 (OECD 414, Oksana N. Khokhlova (2021) BTL BIBC RAS). 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. 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.

After the oral administration of 400 mg/kg bw. first adaptive signs of toxicity were seen with the adult animals. As consequence first signs of reprotoxicity were also seen in this dose group. Therefore this is not a specific reprotoxicity but is caused by the toxic damage of the adults.

5. ASC plus is nearly completely eliminated from serum within 24 hours after administration. There is no potential for accumulation in the body (OECD 417, Zeljenkova, D (2013c)).


6. ASC plus seems to be excreted mainly as unchanged substance via the urine (OECD 417, Zeljenkova, D (2013c))


7. This is in good accordance with the data from ecotoxicity studies showing a slow but steady degradation together with a low toxicity.

In summary the present studies seem to be sufficient for the toxicological evaluation of ASC plus.

Effects on developmental toxicity

Description of key information

The following information is taken into account for any hazard / risk assessment:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male and female rats (Zeljenkova, D (2013)). Treatment of males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period), treatment of females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period) for 54 days.

Additional satellite groups of 5 male and 5 female were treated with 0 and 1600 mg ASC plus/kg bw. and used for observation of reversibility, persistence or delayed occurrence of systemic toxic effects for at least 14 days post treatment.

The test-article was formulated in drinking water and administered in 10 ml/kg bw.

At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOAEL).

1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.

overall NOAEL: 400 mg/kg bw.

Zeljenkova, D (2013):

NOAEL for fertility effects: 1600 mg/kg bw.

NOAEL for developmental effects: 400 mg/kg bw.

 

OECD TG 414 Prenatal Developmental Toxicity Study:

Daily dosages of  0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of   24-25 female Sprague-Dawley rats  from day 5 to day 19 post-mating (inclusive). ASCplus® was formulated in 10 ml/kg bw. destilled water (Oksana N. Khokhlova (2021) BTL BIBC RAS).

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

In the 100 mg/kg bw/day dose group, there were no fetal alterations.

In the 400 mg/kg bw/day dose group the ossification of some bones was delayed.

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.

The NOAEL for maternal toxicity and embryo-fetal development was considered to be 100 mg/kg bw/day.

In summary the present studies seem to be sufficient for the toxicological evaluation of ASC plus.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 MAY 2020 (start of mating) - 29 JAN 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June, 2018

Deviations:

yes

Remarks:

No deviations affecting the data quality, integrity or interpretation were revealed.

GLP compliance:

yes (incl. QA statement)

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 (www.spf-animals.ru)
- Age at study initiation: Approximately 12 weeks old at the initiation of dose administration on gestation day 5
- Weight at study initiation: Body weight at first day of dosing (MEAN ± SD): 213 ± 11 g, N = 99
- Fasting period before study:
- Housing:
Animals were kept in the two corridors barrier-type facility (barrier zone 2 of BTL BIBC RAS).
Cages: 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. The environmental enrichment material Lignocel Nesting Ball (JRS Germany) and red transparent polycarbonate igloo was provided in all cage.
Bedding: Commercial autoclaved woodchip bedding was used (LIGNOCEL BK 8/15, JRS, GmbH). A document from the manufacturer on the composition and quality of the bedding is placed in the study file as raw data. BTL BIBC RAS routinely tests the bedding for microbiological contamination. Results of analyses are kept in an archive at the Test Facility. The copy of the latest check is placed in the study file as raw data
- Diet (e.g. ad libitum): The animals were fed Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaten GmbH, Ferdinand-Gabriel-Weg 16, DE-59494 Soest, Germany) ad libitum. This diet is analyzed by the manufacturer for nutritional components and environmental contaminants and routinely by BTL BIBC RAS for microbiological contaminants. Results of analyses are kept in archive at the Test Facility. The copy of the latest check is placed in the study file as raw data.
- Water (e.g. ad libitum): Filtered tap water was provided ad libitum in standard water bottles. Samples of water are analyzed routinely for microbiological contaminants. Results of analyses are kept in archive at the Test Facility. The copy of the latest check is placed in the study file as raw data.
- Acclimation period: The animals were received from Lab Animals Breeding Center “Pushchino” (Nauki 6, Puschino, Moscow region, Russia 142290 (www.spf-animals.ru) at the age of 4 weeks 07.04.2020 by separate litters to avoid of sibling mating. Each animal was examined on the day of receipt.
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.
- Health: The animal health monitoring is performed by breeder under FELASA-guidelines quarterly in AnLab, s.r.o. (Czech Republic). The result of the last check is attached to the raw data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual mean temperature ranged from 20 °C to 24 °C
- Humidity (%): Mean relative humidity ranged from 30 % to 70 %
There were the deviations from indicated environmental conditions on some dates, which did not negatively influence the animal condition.
- Air changes (per hr): least 12 times hourly
- Photoperiod (hrs dark / hrs light): Automatic change of day and nighttime (08:00-20:00 - "Day", 20:00-08:00 - "Night")

IN-LIFE DATES: From: 30. MAY 2020 To: 08 JUNE 2020

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in a required volume of the vehicle (distilled water) in order to achieve the homogenous suspension. Formulation suspensions were thoroughly homogenized using a magnetic stirrer and sonication.
Test item formulations were prepared every four days, aliquoted to the required volumes of days of the administration and stored in tightly closed glass jars at room temperature in the dark. For the control group, the required volume per day of distilled water was placed in a labeled jar.
On the day of dosing, the aliquot of each formulation was mixed thoroughly and transferred to the barrier zone.

VEHICLE
- Justification: The test item was suspended in water as in recommended vehicle be considered first. This vehicle was also used in the previously conducted screening toxicity study of the test item according to OECD guideline 422.
- Concentration in vehicle: 10, 40, and 160 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

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

Details on analytical verification of doses or concentrations:

Dose Preparation
The Sponsor provided the test item as a neat solid substance. Calculations for the preparation of formulations were based on the dosage level (100, 400 and 1600 mg/kg bw) and administered volume (10 mL/kg body weight), were documented in Excel spreadsheets, and maintained in the study file as printouts.
The test item was suspended in a required volume of the vehicle (distilled water) in order to achieve the homogenous suspension with the concentrations of 10, 40, and 160 mg/mL. Formulation suspensions were thoroughly homogenized using a magnetic stirrer and sonication.
Test item formulations were prepared every four days, aliquoted to the required volumes of days of the administration and stored in tightly closed glass jars at room temperature in the dark. For the control group, the required volume per day of distilled water was placed in a labeled jar.
On the day of dosing, the aliquot of each formulation was mixed thoroughly and transferred to the barrier zone.

Method
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 of ASCplus® (BTL BIBC study No. 704/20) where linearity, sensitivity (LLOQ), specificity/selectivity, accuracy, precision/repeatability, and formulation stability were assessed.

Stability, Homogeneity and Concentration
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).
Analysis of formulations for homogeneity and concentration during the dosing period was conducted in the test facility (BTL BIBC RAS) at the beginning, in the middle, and at the end of the in-life phase using a validated method.
For homogeneity analysis, quadruplicate samples (approximately 0.1 mL of each) were collected from the top, middle, and bottom strata of each dosing formulation prepared during the study.
For concentration analysis, quadruplicate samples were collected from the middle stratum of each dosing formulation (including the vehicle control group) prepared during the study. Samples collected from the mean stratum for homogeneity analysis used for this purpose.
A pair of quadruplicate samples from each stratum was used for analysis, and the other pair was stored as back-up samples at room temperature in tightly closed flasks, analyzed if necessary based on primary assays to verify concentration and were discarded after the study director's approval of the analytical results.
On some date on analysis, 3-4 samples were taken from each level of 160 mg/mL formulation due to the problematic homogenization of this high concentration and variability of the analyzed concentration between samples.
Acceptance criteria for the formulations analysis are based on the test item in vehicle composition as a suspension. The actual concentration of analyzed samples collected from the mean stratum of formulations should be within the range of 85% - 115% of the target concentration. The acceptance criteria for homogeneity are RSD<15% (for suspensions), with the mean concentration within 85% to 115% of the target concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: Females with clear stages of estrous cycle in vaginal smear were cohabited with a male (avoiding siblings mating), 2:1 until mating.
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage. Each mating female was examined daily on the morning. The day when evidence of mating was identified is termed as gestation day 0 (G0).

Duration of treatment / exposure:

Each female was dosed once a day, at approximately the same time each day at the first half of the day (09:00 – 12:00), from day 5 to day 19 (including) of post mating (G5-G19).
The vehicle and the test item were administered orally by gavage, via an appropriately sized stainless steel ball-tipped dosing cannula connected with a syringe once daily. A separate cannula for each group was used.

Frequency of treatment:

once daily

Duration of test:

From day 5 to day 19 (including) of post mating (G5-G19)

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)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

No. of animals per sex per dose:

control group 24, dosage groups 25 females with confirmed mating

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels 100, 400, and 1600 mg/kg bw/day with 4-fold optimal interval were selected for the current study based on data for conducted screening toxicity study of the test item according to OECD guideline 422.
- Rationale for animal assignment (if not random): Before the beginning of the treatment period, the females with confirmed mating were allocated to the experimental groups, according to a stratification procedure, so that the average body weight of each group did not statistically differ. Females with the same day of gestation were allocated to a different group.
- Fasting period before blood sampling for (rat) dam thyroid hormones: Animals were not fasted prior to blood collection.
- Time of day for (rat) dam 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.

Maternal examinations:

CAGE SIDE 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.
- Cage side observations checked: 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.

DETAILED CLINICAL OBSERVATIONS: No

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).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
On gestation day 20, all females were euthanized by anesthesia (Telazol® Zoetis, Spain / Xyla®, i.m.) followed by terminal blood sampling for hormones assay and subjected to hysterectomy.
The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus). Uterus were examined. Ovaries were examined to determined number of corpora lutea.
Each dam was examined macroscopically, thyroid gland was collected in 10% neutral formalin (in complex with trachea and esophagus) and weighed after fixation.

OTHER:
Estrus Cycle Evaluation, Mating and Confirmation of Pregnancy:
After identification, females were monitored to an estrous cyclicity daily during 3-8 days. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage. Each mating female was examined daily on the morning.

T4, T3 and TSH Assay:
Thyroid hormones (thyroxin (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH)) were assayed in serum from all pregnant females (with at least one fetus) by competitive inhibition enzyme immunoassay technique using relevant ELISA kits (see below) and Multiskan™GO Microplate Spectrophotometer (Termo Scientific) and according to standard procedure of manufacturer and SOP of BTL BIBC RAS.

Microscopic Examination of Thyroid Gland:
Thyroid gland of all females euthanized at the scheduled necropsy was trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (the weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus))
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: No
- Serum: Yes
- other:

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
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes
- Anogenital distance of all live rodent pups: Yes

For more details, please refer to 'Any other information'

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. Descriptive statistics (mean, standard deviation (S.D.), and N) are presented for all measurement data and shown in the summary tables. 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.

Historical control data:

historical control data of uterine content
historical control data of body weights, anogenital distances and sex of fetuses
historical control data of fetuses external observations
historical control data of fetal soft tissue examination
historical control data of fetal skeletal and cartilage examination

Clinical signs:

no effects observed

Description (incidence and severity):

No test item related clinical findings were revealed in any dose groups.
In one female (No.24) from the control vehicle group, the focal alopecia was recorded from the gestation day 10 to the day of scheduled euthanasia.
(Tabular summary of the results, see attachment)

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There was no morbidity and mortality of females caused by the test item administration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of pregnant females administered with the 400 and 1600 mg/kg bw/day doses was less compared to the control vehicle group (respectively, by 3.7 % and 4.0 %) to the end of the dosing period (see Table S4, Figure 1A in the attachment). The body weight gain decreased in 1600 mg/kg bw/day dose group starting GD8 and in 400 mg/kg bw/day dose group starting from GD14 (see Figure 1B in the attachment). To the GD20, the body weight gain in these groups was decreased by 5.7 % and 5.8 % compared to the control group.

The gravid uterus in the 400 and 1600 mg/kg bw/day dose groups was less compared to the control value on 10.4 % and 10.9 %, respectively. The final maternal body weight without a gravid uterus did not statistically differ from the body weight of control females (see Table S5 in the attachment).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption in the 100, 400, and 1600 mg/kg bw/day dose treated groups was similar to that in the vehicle control group during all study days.
(Tabular summary of the results, see attachment)

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 treated group.

Histological alterations in the thyroid gland, which were considered to be associated with the test item treatment, were observed in few females of the 1600 mg/kg bw/day dose group. The degeneration of follicular epithelium of minimum grade was found in two females, and C-cell hyperplasia of the slight grade was observed in one female.

ASCplus® lowered thyroid hormone levels in pregnant females. The decrease in T3 was dose-dependent and statistically significant compared to the control value (1.613 ± 0.196 ng/mL) in the 400 mg/kg bw/day dose group (1.496 ± 0.208 ng/mL, p < 0.05) and 1600 mg/kg bw/day dose group (1.485 ± 0.197 ng/mL, p < 0.01). The decrease in T4 level was observed in the 1600 mg/kg bw/day dose group (32.50 ± 3.28 ng/mL versus 34.41 ± 2.57 ng/mL in the control group, p < 0.05). Associated increase in the mean value of TSH in the 1600 mg/kg bw/day dose group (0.389 ± 0.285 µIU/mL versus value 0.315 ± 0.268 µIU/mL in the control group) was not statistically significant; there were no indications of TSH-mediated thyroid gland activation.

For more details on the interpretation of the results, see 'Any other information'; Tabular summary of the results, see attachment.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The absolute and relative thyroid weight of pregnant females was not significantly changed in all dose test item treated group.
(Tabular summary of the results, see attachment)

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.
(Tabular summary of the results, see attachment)

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. 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.
(Tabular summary of the results, see attachment)

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic observations in thyroid glands (please refer to endocrine findings)

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

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.
For more details on the interpretation of the results see 'Any other information'; Tabular summary of the results, see attachment.

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 value of implantation sites per uterus was slightly decreased by 8.6 % (11.7 ± 2.7) and 7.0 % (11.9 ± 2.8), respectively, in the 400 and 1600 mg/kg bw/day dose groups compared to the control group (12.8 ± 1.4).

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 13.5 % in the control vehicle group, p < 0.01, and compared historical reference value 17.8 % for the 400 mg/kg bw/day dose group (p < 0.05)).

The increase in the percentage of pre-implantation loss and associated decrease in the mean number of fetuses per uterus noted for 400 and 1600 mg/kg bw/day dose groups were slight and not dose-dependent; however, the possibility of a test-item relation of this finding cannot be excluded.

For more details on the interpretation of the results, see 'Any other information'; Tabular summary of the results, see attachment.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No litter losses by resorption were observed.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 1600 mg/kg bw/day dose group: slight non-significant increase in the percentage of early resorptions (6.6 % versus 3.6 % in the control vehicle group and 3.4 % as historical control value).

For more details on the interpretation of the results see 'Any other information'; Tabular summary of the results, see attachment.

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:

100 mg/kg bw/day:
No significant findings observed

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)
Implantation: reduced number of implantation sites (by 8.6 % non-significantly); increase in percentage of pre-implantation loss (by 23.3 %, p <0.01)
Thyroids: Decrease in serum T3 level (by 7.3 %, 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)
Implantation: reduced number of implantation sites (by 7.0 % non-significantly); increase in percentage of pre-implantation loss (by 21.5 %, p <0.01)
Thyroids: Decrease in serum T3 level (by 7.9 %, p < 0.01), decrease in serum T4 level (by 5.6 %, p < 0.05), increase in serum TSH level (by 23.5 %, non-significantly)
Thyroids: degeneration of follicular epithelium, minimal grade

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

endocrine findings

pre and post implantation loss

other: Weight change of gravid uterus

Fetal body weight changes:

effects observed, treatment-related

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 a decrease in the male body weight was observed (by 3.6 % ).
(Tabular summary of the results, see attachment)

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

No dead fetuses were recorded.
Difference in mean number of fetuses per animal is mainly associated with a change in implantation.
(Summary in TABLE S9-1, see attachment)

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)
(Tabular summary of the results, see attachment)

Changes in litter size and weights:

not examined

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). Note: The increase in the normalized AGD in female fetuses exposed to 1600 mg/kg bw/day dose is supposed to be caused by the lower fetal body weight and not by the potential hormonal activity of the test item.

(For more details on the interpretation of the results, see 'overall remarks'; Tabular summary of the results, see attachment)

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test item related external malformations were revealed in all dose groups.
Other external observations: see 'Any other information'; Tabular summary of the results, see attachment.

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). 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.
(Tabular summary of the results, see attachment)

Visceral malformations:

effects observed, non-treatment-related

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.
(Tabular summary of the results, see attachment)

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The fetal incidence of malpositioned (undescended) testis was non-significantly increased in the 100 and 1600 mg/kg bw/day dose group compared to the control vehicle group (11.3 % and 11.0 % versus 6.7 %). If compared to the historical control value (3.1 %), these changes were statistically significant (p < 0.05, Fisher exact two-tailed test). However, in these groups, the litter incidence and percentage mean value of affected fetuses per litter were not statistically different. Moreover, the incidence of testes malposition in the 400 mg/kg bw/day dose group was the same as the value in the control group and did not correlate to the decrease in male ratio.
So, the change in the fetal incidence of undescended testes in the low and high dose groups considered to be not test item related.
(Tabular summary of the results, see attachment)

Details on embryotoxic / teratogenic effects:

100 mg/kg bw/day:
No significant findings observed

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)
% males per litter: decreased (45.3 % versus 50.7 % in the control group, p < 0.05)

1600 mg/kg bw/day:
Implantation loss: increase in early resorptions (6.6 % versus 3.6 % in the control group, non-significant)
Fetal body weight: decreased (by 5.0 % for female, p < 0.05, and by 3.6 % for male, non-significantly)
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)
% males per litter: decreased (44.4 % versus 50.7 % in the control group, p < 0.05)

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in sex ratio

fetal/pup body weight changes

other: Skeletal variations

Abnormalities:

not specified

Developmental effects observed:

not specified

Test Item Stability in Formulation

The stability of the test item in the vehicle (water) prepared at concentrations of 10 and 160 mg/mL was checked following 7 days of storage at room temperature (20 - 25 °C) during analytical method validation study.
It was concluded that the formulations of the test item ASCplus in a vehicle (suspension in water) are stable for at least seven days of storage at room temperature (20 - 25 °C). Some deviations in actual concentration are assumed to be caused by the difficulties of the test item homogenization in water.

 

Dose Concentration and Homogeneity (Tabular summary of the results, see attachment)

The homogeneity assessment of the ASCplus dose formulations of 10 and 40 mg/mL met the BTL BIBC SOP requirement, i.e., the RSD for the mean concentration for all strata was ≤15% at a concentration within 85% to 115% of the target level. The variability of concentration for high dose formulation exceeds the acceptable level (RSD = 15.3 %) at the first date of analysis. During re-analysis and in the following dates, the homogeneity of this formulation was acceptable with RSD values of 3.6 – 12.3 %. However, in the first dates of the analysis, the mean actual concentration of 160 mg/mL formulation was less of the acceptable target value (75.5 – 78.0 % of the target concentration). For the last date of analysis, the mean concentration for all strata meat the acceptable level.
The analyzed concentration for 10 and 40 mg/mL formulations was within the acceptable range of 85-115 % of the target concentration at the beginning, middle, and end of the in-life phase of the study. The actual concentration of the test item in the 160 mg/mL formulation was less of the acceptable target value by 4.7 % on date 30.05.2020 (on re-analysis data in the beginning of in-life phase) and by 10.1 % on date 08.06.2020 (end of-in-life phase). On the last date of analysis, the actual concentration of the test item in 160 mg/mL formulation was low but within in acceptable range (89.9 % of taget value).
It is assumed that deviations in 160 mg/mL concentration are due to the difficulty of homogenizing a high dose formulation of the test item insoluble in water as well as to the small increase in the volume of the finished formulation (approximately by 4 %). Based on the formulation analysis, the mean actual concentration of the high dose formulation was approximately 129 mg/mL for the all administration period. No test item was detected in the analyzed vehicle administered to the control group (Group 1).

 

Number of Corpora Lutea

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 attached Table S9-2), but was also statistically significant in the 400 mg/kg bw/day dose group (p < 0.05, Yates corrected Chi-square 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.

 

Uterine Content

The mean number of fetuses per animal was slightly decreased in 400 and 1600 mg/kg bw/day dose groups (by 9.8 % and 5.7 %, respectively, compared to the control vehicle group). This change was not statistically significant but correlated to the slight decrease in the number of implantation sites per animal discussed above and assumed to be test item related. Besides, the number of fetuses in the 400 mg/kg bw/day dose group was lower than the historical control value (see attached Table S9-2, p < 0.05).
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.
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.

 

Weight of Thyroid Glands

The absolute and relative thyroid weight of pregnant females was not significantly changed in all dose test item treated group.

 

Microscopic Observations in Thyroid Glands

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.

 

Thyroid Hormons Assay Data

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 non-significant on 1600 mg/kg bw/day dose without apparent changes in thyroid glands

 

Fetuses External Observation Data

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 lowand 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 data also 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.

 

Undescended Testes Data

The fetal incidence of malpositioned (undescended) testis was non-significantly increased in the 100 and 1600 mg/kg bw/day dose group compared to the control vehicle group (11.3 % and 11.0 % versus 6.7 %). If compared to the historical control value (3.1 %), these changes were statistically significant (p < 0.05, Fisher exact two-tailed test). However, in these groups, the litter incidence and percentage mean value of affected fetuses per litter were not statistically different. Moreover, the incidence of testes malposition in the 400 mg/kg bw/day dose group was the same as the value in the control group and did not correlate to the decrease in male ratio.
So, the change in the fetal incidence of undescended testes in the low and high dose groups considered to be not test item related.

 

Fetal Soft Tissue Examination Data

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.

 

Fetal Skeletal and Cartilage Examination Data

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.

Conclusions:

This study was designed to evaluate the potentially toxic effects of the test item 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid (ASCplus®, CAS 78521-39-8) on pregnancy and embryo-fetal development in rats when administered at doses 100, 400 and 1600 mg/kg bw/day to pregnant rats from implantation (post-mating day 5) to the post-mating day 19 inclusive.
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.

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.

Executive summary:

This prenatal developmental toxicity study (OECD 414, GLP) was conducted to evaluate the potential effects of the test item, 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid (ASCplus®, CAS 78521-39-8), on pregnancy and on embryo-fetal development in rats following daily oral (gavage) administration at doses 100, 400 and 1600 mg/kg body weight/day from implantation to the day prior to scheduled caesarean section (day 5 to day 19 post-mating inclusive).

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.
The fetal findings were described according to the harmonized terminology of the International Federation of Teratology Societies (IFTS) and classified as malformations or variations. Fetal incidence, litter incidence, and affected fetuses per litter were calculated for external, visceral, and skeletal alterations. The reproductive tract was examined with particular attention, and external sex was compared with internal (gonadal) sex in all fetuses. In addition, male fetuses were examined for undescended testes.

 

Results

There was no morbidity and mortality of females caused by the test item administration. No test item related clinical findings were revealed in any dose group.
The body weight of pregnant females in the 400 and 1600 mg/kg bw/day dose groups was slightly decreased compared to the control vehicle group. To the gestation day 20, the body weight in these groups was less (p < 0.05), respectively, by 3.7 % (309 ± 19 g) and 4.0 % (308 ± 21 g) compared to the control group (321 ± 22 g). The body weight gain for the entire period of the test item administration (gestation day 5 to gestation day 20) in these groups was reduced (p < 0.05), respectively, to 44.8 ± 6.6 % (from 213 ± 10 g to 309 ± 10 g) and 44.7 ± 8.4 % (from 213 ± 11 g to 308 ± 21 g) compared to 50.5 ± 5.5 % (from 214 ± 13 g to 321 ± 22 g) in the control group.
Mean food consumption in the 100, 400, and 1600 mg/kg bw/day dose treated groups was similar to that in the vehicle control group during all study days. The lower weight gain of pregnant females in the medium and high dose groups is considered to be due to the lower weight of the gravid uterus. The final maternal body weight in these groups without a gravid uterus did not statistically differ from the body weight of control females, whereas the gravid uterus was less (p < 0.05) compared to the control value (69.0 ± 8.4 g) on 10.4 % (61.8 ± 13.4 g) and 10.9 % (61.5 ± 13.8 g), respectively.

During necropsy, there were no gross findings related to the test item administration. 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 value of implantation sites per uterus was slightly decreased by 8.6 % (11.7 ± 2.7) and 7.0 % (11.9 ± 2.8), respectively, in the 400 and 1600 mg/kg bw/day dose groups compared to the control group (12.8 ± 1.4). 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 13.5 % in the control vehicle group, p < 0.01, and compared historical reference value 17.8 % for the 400 mg/kg bw/day dose group (p < 0.05)). The reduced number of implantation sites in these groups correlated to the slight decrease in number of fetuses per animal (11.1 ± 2.7 and 11.6 ± 2.7, respectively). This change was non-significant compared to the control vehicle group (12.3 ± 1.5); however, it was lower of historical control value (12.7 ± 2.7) for 400 mg/kg bw/day dose (p < 0.05). The increase in the percentage of pre-implantation loss and associated decrease in the mean number of fetuses per uterus noted for 400 and 1600 mg/kg bw/day dose groups were slight and not dose-dependent; however, we cannot deny the test item relation of this finding. Potentially, the first dose of the test item could affect the implantation process or the blastocyst. In rats, the implantation window corresponds to a period of approximately 24 h between day 5 and day 6 of post-mating. The first dose of the ASCplus® was administered about 5.5 days after mating with a variability of the pre-administration period of pregnancy from 5 days 5 hours to 5 days 17 hours. Therefore, it is likely that the first administered dose of 400 and 1600 mg/kg can influence blastocyst or implantation in some females (when dosing happened in the implantation window). Moreover, in the 1600 mg/kg bw/day dose group, a decrease in the mean number of fetuses per animal correlated not only with a decrease in the number of implantations, but also with a slight non-significant increase in the percentage of early resorptions (6.6 % versus 3.6 % in the control vehicle group and 3.4 % as historical control value).

The absolute and relative thyroid weight of pregnant females was not significantly changed in all dose treated group. Histological alterations in the thyroid gland, which were considered to be associated with the test item treatment, were observed in few females of the 1600 mg/kg bw/day dose group. The degeneration of follicular epithelium of minimum grade was found in two females, and C-cell hyperplasia of the slight grade was observed in one female. ASCplus® lowered thyroid hormone levels in pregnant females. The decrease in T3 was dose-dependent and statistically significant compared to the control value (1.613 ± 0.196 ng/mL) in the 400 mg/kg bw/day dose group (1.496 ± 0.208 ng/mL, p < 0.05) and 1600 mg/kg bw/day dose group (1.485 ± 0.197 ng/mL, p < 0.01). The decrease in T4 level was observed in the 1600 mg/kg bw/day dose group (32.50 ± 3.28 ng/mL versus 34.41 ± 2.57 ng/mL in the control group, p < 0.05). Associated increase in the mean value of TSH in the 1600 mg/kg bw/day dose group (0.389 ± 0.285 µIU/mL versus value 0.315 ± 0.268 µIU/mL in the control group) was not statistically significant; there were no indications of TSH-mediated thyroid gland activation.
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 % (3.44 ± 0.23 g, p < 0.05) compared to the control vehicle group (3.62 ± 0.37 g), and a tendency to a decrease in the male body weight was observed (3.72 ± 0.25 g compared to 3.86 ± 0.36 g).
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 (respectively, 5.0 ± 1.8 and 5.1 ± 1.7 versus 6.3 ± 1.3, p < 0.05). The percentage of males 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 compared to the historical control value (54.1 % of males).
No evidence of anti-androgenic activity of the test item was found. The fetal incidence of malpositioned (undescended) testis was non-significantly increased in the 100 and 1600 mg/kg bw/day dose group compared to the control vehicle group (11.3 % and 11.0 % versus 6.7 %). If compared to the historical control value (3.1 %), these changes were statistically significant (p < 0.05, Fisher exact two-tailed test). However, in these groups, the litter incidence and percentage mean value of affected fetuses per litter were not statistically different. Moreover, the incidence of testes malposition in the 400 mg/kg bw/day dose group was the same as the value in the control group and did not correlate to the decrease in male ratio. The change in the fetal incidence of undescended testes in the low and high dose groups is considered to be non-treatment related. The absolute and normalized anogenital distance in male fetuses was not significantly changed in all dose groups. As a speculation, a lower number of males in the medium and high dose groups may be associated with a decrease in implantation sites and is caused by a male loss at the initial stage of embryogenesis. In female fetuses, the normalized AGD value was slightly but significantly (p < 0.05) increased in the 1600 mg/kg bw/day dose group (0.73 ± 0.03) compared to the normalized AGD in female fetuses exposed to high dose is supposed to be caused by the lower fetal body weight and not by the potential hormonal activity of the test item.
No test item related external malformations were revealed in fetuses of 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). In total, anasarca was observed in 3 fetuses in the lower dose group (from one litter) and four fetuses from the control vehicle group (from one litter), 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.
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.
The test item did not cause an increase in the incidence of findings in fetal soft tissues. The fetal and litter incidence was approximately the same among all groups, including control, so they are not considered treatment-related.
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), fifth metacarpal (48.5 % versus 36.2, p < 0.05), and total unossified phalanges of forepaws (56.0 % versus 39.6 %, p < 0.01) was noted. The incidence of total affected fetuses in the 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.
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. 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. The disturbances in maternal thyroid hormone homeostasis likely contribute to the reduction in the observed fetal skeletal ossification.

 

Conclusion
This study was designed to evaluate the potentially toxic effects of the test item 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid (ASCplus®, CAS 78521-39-8) on pregnancy and embryo-fetal development in rats when administered at doses 100, 400 and 1600 mg/kg bw/day to pregnant rats from implantation (post-mating day 5) to the post-mating day 19 inclusive.
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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in accordance to OECD guideline

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany, D-97633 Sulzfeld
- Age at purchase: 11 weeks
- weight rage at time of grouping: males, 175-200 g
- Fasting period before study: no
- Housing: 2 per cage,
- Cages: TECHNIPLAST filter top cages type 2145 F with an G-Temp (PSU) durable filter cover, 480x265x210 mm³, floor area 940 cm²,
- Source: Techniplast Company, Italy
- Diet: ad libitum, M3, BONAGRO Ltd., reg. CZ 10174, Czech Republic
- Water: ad libitum, tap water
- Acclimation period: 9 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE: suspension in water
- Amount of vehicle: 10 ml/kg given at similar times each day

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1 (probably) (male animals: see Endpoint study record in section 7.5.1 "Repeated dose Toxicity")
- Length of cohabitation: until copulation/ up to 14 days
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- M/F ratio per cage: 1/1
- Length of cohabitation: Each morning the females were examined for the presence of sperm in vaginal lavages.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually.
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period)
females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period)

Frequency of treatment:

daily by stomach tube

Duration of test:

up to 54 days

Remarks:

Doses / Concentrations:
0, 100, 400, 1600 mg/kg bw./day
Basis:
actual ingested

No. of animals per sex per dose:

12
satellite group: (1600 mg/kg bw./day ):5 individuals (5 females and 5 males)

Control animals:

yes

Details on study design:

- Control groups: drinking water
- Dose selection rationale: Results of an acute toxicity study with oral administration to male and female rats
- Result: no effects up to and including 2000 mg/kg bw.
- Rationale for animal assignment: randomly grouped
- Section schedule rationale: all animals were sacrificed

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on administration days 1, 8, 15, 22, 29, 36, 42, 50 and on day of autopsy

FOOD CONSUMPTION : 2 weeks before mating, after 2 week mating period and weekly until the end of the study

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 14 (before mating) and prior to autopsy from the satellite groups and from the control and high dose group.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:day 14 (before mating) and prior to autopsy from the satellite groups and from the control and high dose group.

URINALYSIS: No

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]

Statistics:

Statistical evaluation was operated using the software SPPS version 16.0. Group data were represented by mean, standard deviation and median. Statistical analysis in case of date measured once during the study (organ weight, haematology, clinical chemistry) : Mann-Whitney U test for pairwise comparison between control and individual experimental groups on significance level alpha = 0.05.
Statistical analysis in cas of repeated data measurement (body weight, food intake): Repeated measures ANOVA (procedure General Linear Model (GLM) for Repeated Measures in SPSS statistical software).

Indices:

implantation/fertility index, delivery index, live birth index, neonatal survival rate, copulation index, lactation status

Historical control data:

no

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

clinical biochemistry

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

other: other:

Dose descriptor:

LOEL

Effect level:

1 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: body weight and mortality

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

LOEL

Effect level:

1 600 other: mg/kg bw/day mother

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At birth no differences were observed between the groups, but on day 4 after birth a higher number of dead pups was counted in the high-dose level group.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male and female rats. Treatment of males: 54 administrations, (2 weeks before mating, 2 weeks during mating and 26 days after mating period), treatment of females: 14 days pre-mating period and until mating, through the pregnancy period (assuming 20 days pregnancy) until lactation day 3 after delivery (4 days lactation period) for 54 days.
Additional satelite groups of 5 male and 5 female were treated with 0 and 1600 mg ASC plus/kg bw. and used for observation of reversibility, persistence or delayed occurence of systemic toxic effects for at least 14 days post treatment.
The test-article was formulated in drinking water and administered in 10 ml/kg bw..
At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOAEL).
1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.
NOAEL: 400 mg/kg bw.

Executive summary:

Daily dosages of 0, 100, 400, and 1600 mg ASC plus/kg bw. were administered by stomach tube to groups of 12 male rats for 54 days. The test-article was formulated in drinking water and administered in 10 ml/kg bw..

Histopathology and gross necropsy did not reveal any test article related changes.

The organ weights showed some statistically significant differences, but these were not related to dosage or not affirmed by the findings in other groups, for example by the results of the satelite groups.

The same is true for the statistical significant differences of the haematological parameters and the result of the clinical chemistry, especially decreased activities of the liver enzymes in the blood. These are merely statistical effects without biological relevance.

Two effects with a possible relation to the oral administration of ASC plus were observed:

- At birth no differences were observed between the groups, but on day 4 after birth a higher number of dead pups was counted in the high-dose level group.

- With male rats a dose dependent decrease in body weight is observed. This effect is in the highest dose group statistically significant and confirmed by the satellite group but not confirmed by the body weight development of the females and not confirmed by the food consumption of males and females.

Although an influence of an infection with paracites seems possible and may explain both effects, a non-specific influence of ASC plus cannot be excluded. The daily administration of a suspension of ASC plus in 10 ml/ kg bw. may have led to a reduced appetite of the animals. This should be especially true for the highest dosage group, as here the suspension was relatively dense. Imaginable is also an osmotic effect of non-resorbed ASC plus in the intestine.At dosages of 100 and 400 mg/kg bw. the animals showed no differences to the control animals (NOEL).

1600 mg ASC plus/kg bw. may have induced an influence on the body weight gain of the males and the survival of pups until day 4 after birth.

With regard to the results of the present study 400 mg ASC plus/kg bw. per day can be defined as "No Observed Adverse Effect Level" (NOAEL) for the developmental toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable guideline studies

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

According to the present data the toxicity and the toxicokinetics of ASC plus (6-[[(4-methylphenyl) sulphonyl]amino]hexanoic acid) can be described as follows:

1. At dosages of 100 mg/kg bw. the animals showed no differences to the control animals (NOAEL) (OECD 414).


2. 400 mg ASC plus/kg bw. (OECD 408) may have induced:

with Males: increased serum urea and Leydig cells hyperplasia in the testes,

With Females: decreased APTT, increased liver weights; liver, fatty change; kidney, lipofuscinosis, calculi in pelvis; thyroid glands, C-cell hyperplasia.

3. 1600 mg ASC plus/kg bw. (OECD 408) may have induced:

Males, 1600 mg/kg bw/day: increased urobilinogen, decreased urine pH, decreased serum triglyceride, increased serum urea, increased liver weights and kidney weights; liver, fatty change; kidney, lipofuscinosis; thyroid glands, C-cell hyperplasia; testes, Leydig cells hyperplasia;

Females, 1600 mg/kg bw/day: increased urobilinogen, decreased: APTT, increased liver weights and kidney weights; liver, fatty change, hepatocellular hypertrophy; thyroid glands, C-cell hyperplasia.

4. 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 (OECD 414, Oksana N. Khokhlova (2021) BTL BIBC RAS). 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. 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.

After the oral administration of 400 mg/kg bw. first adaptive signs of toxicity were seen with the adult animals. As consequence first signs of reprotoxicity were also seen in this dose group. Therefore this is not a specific reprotoxicity but is caused by the toxic damage of the adults.

5. ASC plus is nearly completely eliminated from serum within 24 hours after administration. There is no potential for accumulation in the body (OECD 417, Zeljenkova, D (2013c)).


6. ASC plus seems to be excreted mainly as unchanged substance via the urine (OECD 417, Zeljenkova, D (2013c))


7. This is in good accordance with the data from ecotoxicity studies showing a slow but steady degradation together with a low toxicity.

In summary the present studies seem to be sufficient for the toxicological evaluation of ASC plus.

Justification for classification or non-classification

There was no treatment related effect that would fullfill any criteria for classification and labelling according to the criteria laid out in

Regulation (EC) No.: 1272/2008 (especially 3.7 "Reproductive toxicity" and "3.9. Specific target organ toxicity — repeated exposure"). Also, according to the criteria specified by Directive 67/548/EEC the substance is not classified.

Justification:

In the 90 days repeated toxicity study the females showed clear maternal toxicity after daily dosages of 400 mg/kg bw.: decreased APTT, increased liver weights; liver: fatty change; kidney: lipofuscinosis, calculi in pelvis; thyroid glands: C-cell hyperplasia. At this dosage in an OECD 414 Prenatal Developmental Toxicity Study the fetuses only showed delayed ossification of some bones. Based on the 90 day toxicity study OECD 408, this change in the fetuses is clearly a result of systemic toxicity to the female mother animals and does not indicate a specific developmental toxicity.

After the oral administration of 400 mg/kg bw. in the OECD 414 study first adaptive signs of toxicity were seen with the adult animals. As consequence first signs of reprotoxicity were also seen in this dose group. Therefore, this is not a specific reprotoxicity but is caused by the toxic damage of the adults.

After daily dosages of 100 mg/kg bw. for 90 days no toxic changes were seen with the female animals (OECD 408). The OECD 414 Prenatal Developmental Toxicity Study also showed no fetal alterations after daily dosages of 100 mg/kg bw.

By analogy with the classification rules of the repeated dose toxicity the no-effect dosage of 100 mg/kg bw is above the criteria for classification and labelling. The next higher dose (400 mg/kg bw), however, showed the delayed ossification of bones in the fetuses only when maternal toxicity occurred. A classification is only necessary in the absence of maternal toxicity (CLP regulation). So the criteria for classification and labelling are not fulfilled. Therefore, no classification and labelling is required.