Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction
Remarks:
other: screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 422. The study was conducted on 6-[(p-Tosyl)amino]hexanoic acid, which is the carboxylic acid component of the registered substance i.e. tosyl salt.
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 (incl. QA statement)
Limit test:
no
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
Description (incidence and severity):
f
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)
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Considered to be the NOAEL for systemic and reproduction-developmental toxicity.
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 were 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 statistically 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.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: 1600 mg test substance may have induced an influence on the body weight development of the males and the survival of pups until day 4 after birth.
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 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.
NOAEL: 400 mg/kg bw. However NOAEL (Fertility) = 1600 mg/kg bw.
Executive summary:

The toxicity of the test substance to reproduction was determined in accordance with the OECD Guideline for Testing of Chemicals 422. Dose of 0, 100, 400, and 1600 mg/kg bw/day of the test substance 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 the test substance 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 the influence of an infection by a parasite could be a possible explanation of both effects, a non-specific influence of the test substance cannot be excluded. The daily administration of a suspension of the test substance in 10 ml/ kg bw may have led to a reduced appetite of the animals. This could be especially true for the highest dosage group, as here the suspension was relatively dense. It is also possible that there was an osmotic effect of non-resorbed test substance in the intestine. At dosages of 100 and 400 mg/kg bw the animals showed no differences to the control animals (NOEL). a dose of 1600 mg/kg bw/day of the test substance 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/kg bw/day of the test substance can be defined as "No Observed Adverse Effect Level" (NOAEL). The NOEL and NOAEL for systemic toxicity in the parent generation of test animals was found to be 400 mg/kg bw/day for both.

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

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study results were published in a peer reviewed journal.
Qualifier:
no guideline followed
Principles of method if other than guideline:
No information was provided to indicate which specific guideline was followed.
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
dermal
Vehicle:
acetone
Details on exposure:
The substance was applied dermally to the interscapular area of the clipped back.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
10 weeks
Frequency of treatment:
Daily
Details on study schedule:
Application was made during breeding, and through gestation and lactation for females.
Remarks:
Doses / Concentrations:
1.8 ml/kg
Basis:
no data
No. of animals per sex per dose:
No data
Clinical signs:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
No effects on mating or fertility
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified
No effects on offspring growth or survival
Reproductive effects observed:
not specified
Conclusions:
The substance produced no effects on mating, fertility, or offspring growth and survival.
Executive summary:

The reproductive toxicity of the substance was determined via dermal exposure to mice in a mating study. 0.5 g/kg of triethanolamine in acetone was applied dermally to the interscapular area of the clipped back in an approximate volume of 1.8 ml/kg daily for 10 weeks prior to mating, during breeding, and through gestation and lactation for females. The substance had no effect on mating, fertility, or offspring growth and survival.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 600 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat
Additional information

The toxicity of the carboxylic acid component of the registered substance (tosyl salt) to reproduction was determined in accordance with the OECD Guideline for Testing of Chemicals 422. Dose of 0, 100, 400, and 1600 mg/kg bw/day of the test substance 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 the test substance 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 the influence of an infection by a parasite could be a possible explanation of both effects, a non-specific influence of the test substance cannot be excluded. The daily administration of a suspension of the test substance in 10 ml/ kg bw may have led to a reduced appetite of the animals. This could be especially true for the highest dosage group, as here the suspension was relatively dense. It is also possible that there was an osmotic effect of non-resorbed test substance in the intestine. At dosages of 100 and 400 mg/kg bw the animals showed no differences to the control animals (NOEL). a dose of 1600 mg/kg bw/day of the test substance 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/kg bw/day of the test substance can be defined as "No Observed Adverse Effect Level" (NOAEL). The NOEL and NOAEL for systemic toxicity in the parent generation of test animals was found to be 400 mg/kg bw/day for both.


 


Read-across between the tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered substance is considered justified as the registered substance is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid by simple neutralisation with triethanolamine (TEA). Other than ionization of the carboxylic acid group, the 6-[(p-Tosyl)amino]hexanoic acid remains chemically unchanged upon salt formation. In water, the acid and amine components of 6-[(p-Tosyl)amino]hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) dissociate completely and behave essentially as independent substances. Since TEA can be considered non-hazardous, it is the acid component of the salt that will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the carboxylic acid group in 6-[(p-Tosyl)amino]hexanoic acid (pKa = 4.90) is the same in the free acid as it is in the TEA salt. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the same way whether it is in the salt form or as the parent carboxylic acid and hence it’s bioavailability will be the same. In addition, the triethanolamine component of the registered substance was found to not be toxic to reproduction. As such, the registered substance (tosyl salt) is not considered to be toxic to reproduction and does not need to be classified as such according to EU CLP criteria.


 


Short description of key information:


The toxicity of the carboxylic acid component of the registered substance (tosyl salt) to reproduction was determined in accordance with the OECD Guideline for Testing of Chemicals 422. With regard to effects on fertility of the present study 1600 mg/kg bw/day of the test substance can be defined as "No Observed Adverse Effect Level" (NOAEL). The NOEL and NOAEL for systemic toxicity in the parent generation of test animals was found to be 400 mg/kg bw/day for both. In addition, the triethanolamine component of the registered substance was found to not be toxic to reproduction.


 


Justification for selection of Effect on fertility via oral route:


Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 422. The study was conducted on the carboxylic acid component of the registered substance (tosyl salt).


 


Justification for selection of Effect on fertility via inhalation route:


This study is not deemed necessary as the substance does not solidify readily and when it does it is a waxy solid and does not form inhalable particles. The substance has also been estimated to have a very low vapour pressure via modelling using the EPIsuite modelling program. The substance is provided to customers only in formulated products and is not isolated. Therefore, inhalation is not considered to be a significant route of exposure to the registered substance, in accordance with REACH Annex XI, Column 2. The results of a repeated dose oral toxicity study will be provided (section 7.5.1). It is therefore considered unjustified to perform further animal testing.


 


Justification for selection of Effect on fertility via dermal route:


Results for the triethanolamine component of the registered substance show  that the test substance produced no effects on mating, fertility, or offspring growth and survival.

Effects on developmental toxicity

Description of key information

In the available OECD 414 toxicity study with carboxylic acid component of the registered substance (tosyl salt), in which the substance was tested at 100, 400, and 1600 mg/kg bw/day, some findings in fetuses were related to altered ossification; however, matemal thyroid hormone homeostasis likely contributed to those effects. In the same study, there was also a decrease in percent of males per litter and although the effects were statistically significant, the effect levels were low and did not show an increase in incidence between 400 and 1600 mg/kg bw/day. Moreover, in the availble OECD 422 screening study, there was no effect on number of males in the litters on PND 0. Overall, availble studies do not indicate any signs of developmental toxicity with the test susbtance. 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Remarks:
Source study for the purpose of read-across to the registered substance
Adequacy of study:
key study
Study period:
2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 414. The study was conducted on 6-[(p-Tosyl)amino]hexanoic acid, which is the carboxylic acid component of the registered substance i.e. tosyl salt.
Justification for type of information:
The test substance was the carboxylic acid component of the registered substance (tosyl salt). Readacross between the tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered
substance is considered justified as the registered substance is manufactured directly from 6-[(pTosyl)amino]hexanoic acid by simple neutralisation with triethanolamine (TEA). Other than ionization of
the carboxylic acid group, the 6-[(p-Tosyl)amino]hexanoic acid remains chemically unchanged upon salt
formation. In water, the acid and amine components of 6-[(p-Tosyl)amino]hexanoic acid, compound with
2,2’,2’’-nitrilotriethanol (1:1) dissociate completely and behave essentially as independent substances.
Since TEA can be considered non-hazardous, it is the acid component of the salt that will have a
more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the
carboxylic acid group in 6-[(p-Tosyl)amino]hexanoic acid (pKa = 4.90) is the same in the free acid as
it is in the TEA salt. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the
same way whether it is in the salt form or as the parent carboxylic acid and hence it’s bioavailability
will be the same.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS:Nauki 6, Puschino, Moscow region, Russia 142290
- Age at study initiation: Approximately 12 weeks old at the initiation of dose administration on gestation day 5
- Weight at study initiation: 213 ± 11 g, N = 99
- Fasting period before study: Food consumption was assessed for each female quantitatively as g/kg of body weight/day by weighing of feeder (cage lid) at the beginning of the day and 24 hours after. Food consumption was recorded prior to the initiation of dose administration (gestation days 0-1), and at three-day intervals thereafter (gestation days 4-5, 7-8, 10-11, 13-14, 16-17 and 19-20).
- Housing: All animals were housed in solid bottom polycarbonate cages (Type IV, 598 х 380 х 200 mm (LxWxH), 2272 cm sq., Tecniplast s.p.a.) with bedding. Cages are equipped with steel lids, steel separators for the food and steel label holders. After identification and until mating, all females were housed by groups. Males were housed alone, and females cohabited with a male in the home cage of the male (2:1). After confirmed mating, dams were housed alone until euthanasia on gestation day 20 (G20).
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: During adaptation/acclimatization, animals were kept in groups (by litter/sex) in the barrier zone of facility and animal’s condition was evaluated daily by cageside observation. Before pre-mating oestrus cycle evaluation, all animals were identified by ear punch, weighed, and clinical observations were recorded. For all males, the testes were palpated, and for all females, the opened vagina was inspected visually. Animals considered unsuitable for the study were excluded prior to mating.

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

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

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

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




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

The stability of the test item in the vehicle (water) prepared at concentrations of 10 and 160 mg/mL was confirmed following storage for 7 days at room temperature (the temperature range, 20 – 25 °C) during method validation study. Besides, homogeneity analysis was performed for formulations of 10 and 160 mg/mL after 4 days of storage after re-mixing. Results on formulation stability are presented in the report on analytical method validation (in the study 704/20 report).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: until evidence of mating by vaginal copulatory plug or the presence of sperm following a vaginal lavage.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
From day 5 to day 19 post-mating (days G5-G19)/ Three groups received the test item at dose-levels of 100, 400 or 1600 mg/kg bw/day.
Frequency of treatment:
Once daily
Duration of test:
From implantation to the day prior to scheduled caesarean section (day 5 to day 19 post-mating inclusive). On day 20 post-mating, the dams were sacrificed.
Dose / conc.:
1 600 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 groups of 24-25 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels 100, 400, and 1600 mg/kg bw/day with 3-4-fold optimal interval were selected for the current study based on data for conducted screening toxicity study of the test item according OECD guideline 422.

All females were observed twice daily for mortality and morbidity and for signs of toxicity following dose administration. Body weights and food consumption are recorded at three-day intervals. On day 20 post-mating, the dams were sacrificed and subjected to a macroscopic examination and enumeration of corpora lutea. The weight of the thyroid gland, histopathological assessment of the thyroid gland, and assay of serum concentration of thyroxin (T4), triiodothyronine (T3), and thyroid stimulating hormone (TSH) were done in dams to observe pathological changes in thyroid function. Gravid uteri were weighed, and uteri content are examined to record implantation sites, early and late resorptions, dead, and live fetuses. The fetuses were weighed, sexed with measurement of anogenital distance (AGD), and submitted to external examination. Approximately half of the fetuses from each litter were subjected to a detailed examination of soft tissue by serial sectioning after fixation in Bouin’s solution while the other half underwent detailed skeletal examination following staining of bone with alizarin red and cartilage with alcian blue.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon at the same time, for morbidity and mortality. Each female was also observed for signs of toxicity approximately 30-45 minutes following dose administration. In addition, the presence of findings at the time of dose administration was recorded for individual animals.

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

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: A complete necropsy was conducted on all females at scheduled termination. Necropsy included examination of the external surface of the body, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal and pelvic cavities including viscera.
Hysterectomy and examination of uterine content was done for all females. Ovaries were examined to determined number of corpora lutea. Gravid uterine weight was collected at scheduled necropsy. Uteri, which appear non-gravid by macroscopic examination, were opened and placed in 10 % ammonium sulfide solution for detection of early implantation loss. Thyroid glands were preserved and weighed after fixation.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: AGD, mm, divided to the cube root of the body weight
Statistics:
All statistical tests were performed using Microsoft Excel (descriptive statistics) and statistical software Statistica for Window v.7.1 to compare the treated groups to the control group. The litter is accepted as an experimental unit for statistical analysis.

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

Descriptive data, percentage values, and pathomorphological data were analyzed by Fisher's Exact Test; the percentage of pre-implantation loss was analyzed by Yates' corrected Chi-square test.
Indices:
Former implantation sites, number of corpora lutea, implantation loss
Historical control data:
Historical control data were used for:
-UTERINE CONTENT
-BODY WEIGHTS, ANOGENITAL DISTANCES AND SEX OF FETUSES
-FETUSES EXTERNAL OBSERVATIONS
-FETAL SOFT TISSUE EXAMINATION
-FETAL SKELETAL AND CARTILAGE EXAMINATION

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
400 mg/kg bw/day
Gravid body weight gain: decrease on 5.7 % (p < 0.05)
Gravid uterine weight: decreased on 10.4 % (p < 0.05)

1600 mg/kg bw/day
Gravid body weight gain: decrease on 5.8 % (p < 0.05)
Gravid uterine weight: decreased on 10.9 % (p < 0.05)
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Behaviour (functional findings):
no effects observed
Gross pathological findings:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
400 mg/kg bw/day
Implantation: reduced number of implantation sites (by 8.6 % non-significantly); increase in percentage of pre-implantation loss (by 23.3 %, p <0.01)

1600 mg/kg bw/day
Implantation: reduced number of implantation sites (by 7.0 % non-significantly); increase in percentage of pre-implantation loss (by 21.5 %, p <0.01)
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
1600 mg/kg bw/day
Implantation loss: increase in early resorptions (6.6 % versus 3.6 % in the control group, non-significant)
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
pre and post implantation loss
other: endocrine findings (migrated information)
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
1600 mg/kg bw/day
Fetal body weight: decreased (by 5.0 % for female, p < 0.05, and by 3.6 % for male, non-significantly)
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
400 mg/kg bw/day
% males per litter: decreased (45.3 % versus 50.7 % in the control group, p < 0.05)

1600 mg/kg bw/day
% males per litter: decreased (44.4 % versus 50.7 % in the control group, p < 0.05)
Changes in postnatal survival:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
400 mg/Kg bw/day
Skeletal variations – Forelimb: increase in fetal incidence of incomplete ossification in 5th metacarpal (p < 0.05)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in interparietal bone (p < 0.05)

1600 mg/kg bw/day
Skeletal malformation: Sternum – split (one fetus)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in frontal bone (p < 0.05)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in interparietal bone (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of incomplete ossification in 5th metacarpal (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of unossified phalanges (p < 0.01)
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull sutures
skeletal: forelimb
Description (incidence and severity):
400 mg/kg bw/day
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in interparietal bone (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of incomplete ossification in 5th metacarpal (p < 0.05)

1600 mg/kg bw/day
Skeletal malformation: Sternum – split (one fetus)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in frontal bone (p < 0.05)
Skeletal variations - Skull: increase in fetal incidence of incomplete ossification in interparietal bone (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of incomplete ossification in 5th metacarpal (p < 0.05)
Skeletal variations – Forelimb: increase in fetal incidence of unossified phalanges (p < 0.01)
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Conclusions:
Daily dosages of 100, 400, and 1600 mg/kg bw of the test substance were administered by gavage once daily to three groups of Sprague-Dawley female rats from day 5 to day 19 post-mating (inclusive). A concurrent vehicle control group received the vehicle (distilled water) on a comparable regiment and in the same volume of 10 mL/kg bw. Each group consists of 24-25 females with confirmed mating.

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

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


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


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



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


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


 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 600 mg/kg bw/day
Study duration:
subacute
Species:
rat
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

The developmental toxicity of the test substance (tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid)) was determined in accordance with the OECD Guideline for Testing of Chemicals 414.


Doses of 0, 100, 400, and 1600 mg/kg bw/day of the test substance were administered by gavage from day 5 to day 19 post-mating to groups of 24-25 females with confirmed mating. The test-article was formulated in drinking water and administered in 10 ml/kg bw.


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. In the 1600 mg/kg bw/day dose group, delayed ossification was observed, which was associated with reduced fetal bodyweight. Delayed ossification in fetuses correlated to the disturbance in matemal homeostasis of thyrotd hormones.


The percentage of males per litter was decreased at both 400 and 1600 mg/kg bw/day dose groups compared to control with no difference observed between 400 and 1600 mg/kg bw/day. Changes in number of males per litter were not observed at PND 0 in the complimentary OECD 422 screening study.


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


 


Justification for selection of Effect on developmental toxicity: via oral route:


Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 414. The study was conducted on the carboxylic acid component of the registered substance (tosyl salt).


 


Justification for selection of Effect on developmental toxicity: via inhalation route:


This study is not deemed necessary as the substance does not solidify readily and when it does it is a waxy solid and does not form inhalable particles. The substance has also been estimated to have a very low vapour pressure via modelling using the EPIsuite modelling program. The substance is provided to customers only in formulated products and is not isolated. Therefore, inhalation is not considered to be a significant route of exposure to the registered substance, in accordance with REACH Annex XI, Column 2. The results of a repeated dose oral toxicity study will be provided (section 7.5.1). It is therefore considered unjustified to perform further animal testing.


 


Justification for selection of Effect on developmental toxicity: via dermal route:


This study is not considered necessary as repeated dose toxicity via the oral route will be provided (section 7.5.1), in accordance with REACH Annex IX, Column 2. Dermal exposure to the substance is not considered likely as the substance is supplied in an aqueous formulation without isolation of the substance. In addition, the substance is directly added to the formulated product without isolation so skin contact is not considered a major exposure route to this substance. Therefore, further animal testing is considered to be unjustified.

Justification for classification or non-classification

Available studies (OECD 414, OECD 422) on the read across substance indicate that classification of target substance is not justified.

Additional information