3-cyclohexylaminopropane-1-sulphonic acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP OECD 421 guideline study without relevant deviations on the registered substance itself.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 421 (1995): “Reproduction/developmental toxicity screening test”

Deviations:

yes

Remarks:

During the experiment, the air temperature exceeded 25ºC a few times, and the relative air humidity exceeded 70% a few times. These changes were temporary and did not influence the study course and results.

GLP compliance:

yes (incl. QA statement)

Remarks:

Bureau for Chemical Substances, Poland

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-cyclohexylaminopropane-1-sulphonic acid

EC Number:

214-492-1

EC Name:

3-cyclohexylaminopropane-1-sulphonic acid

Cas Number:

1135-40-6

Molecular formula:

C9H19NO3S

IUPAC Name:

3-cyclohexylaminopropane-1-sulphonic acid

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 3-(cyclohexylamino)-propane sulfonic acid
- Substance type: pure substance
- Storage condition of test material: in a plastic container, 15-25°C

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: husbandry of laboratory animals of the Experimental Medicine Centre at the Medical University in Białystok
- Age at study initiation: (P) 11 wks
- Weight at study initiation: (P) Males: 347.4±19.92 (group 0), 347.7±15.81 (group 1), 348.2±16.93 (group 3), 347.3±10.59 (group3) g; Females: 223.6±16.01 (group 0), 223.5± 16.03 (group 1), 223.7±15.26 (group 2), 223.7±15.51 (group 3) g; body weight of individual animals was ± 20% of the average value for each sex
- Housing: The animals were kept in plastic cages covered with wire bar lids. The dimensions of the cages were 58 x 37 x 21 cm (length x width x height). During the experiment, there were 3 animals in one cage. Each sex was kept separately. For the purpose of mating, one female was caged with one male. Pregnant females were housed individually. After delivery, they were housed with their offspring.
- Diet (e.g. ad libitum): Murigran standard granulated laboratory fodder produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz (batch numbers: 3/15, 4/15, 5/15, and 6/15) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 26°C
- Humidity (%): 35 - 90%
- Air changes (per hr): 16 times/h
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark (artificial, fluorescent lighting)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item were prepared every day, i.e. directly before the administration.

VEHICLE
- Concentration in vehicle: 100, 400, 1600 mg/mL
- Amount of vehicle (if gavage): 0.5 mL/kg bw
- Purity: distilled

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until pregnancy occurred
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Males in the study were dosed for 28 days (2 weeks prior to mating, during the mating period, and post-mating period). Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and 4 days of lactation (40 - 47 days).

Frequency of treatment:

once a day for seven days a week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 800 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

12 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of the test item administered to the treated groups were determined on the basis of the results of the repeated dose 28-day oral toxicity study on rats.
- Rationale for animal assignment (if not random): The animals were randomized to individual groups. Their sexes and body weights were taken into account.

Positive control:

not required

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The evaluation of the general condition of animals, i.e. the observation of all animals for morbidity and mortality was conducted twice a day or once a day (on days off).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The detailed clinical observations were conducted daily. They involved the evaluation of skin, fur, eye and mucosa changes, the respiratory, circulatory, and autonomous and central nervous systems, somatic activity, and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males were measured twice a week during the entire experiment. Body weights of females were measured twice a week before gestation, on days 0, 7, 14, and 20 of gestation, on day 0 or 1 post-partum, and on day 4 post-partum. Body weights of pups were measured on days 0 or 1 and 4 post-partum

FOOD CONSUMPTION:
Food intake by parental males was measured once a week during the pre-mating period. Food intake by parental females was measured once a week before gestation, on days 0, 7, 14, and 20 of gestation, and on days 0 or 1 and 4 post-partum.
Food intake/cage was measured. It was converted into average food intake/100 g b.w.

OTHER:
The following parameters were also determined:
- the average number of days required for copulation (the day when males and females were caged together was day 0);
- the length of gestation.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight
Detailed examinations of the testicles and epididymides were conducted to determine potential disorders of spermatogenesis. In case of the testicles, spermatogenetic cells (spermatogonia, spermatocytes, and spermatids) were examined. The examination involved the evaluation of the conformation of individual layers of the genital epithelium and the seminiferous tubule lumen content to exclude the presence of immature forms of spermatogenetic cells or giant cells. For the epididymides, the presence of sperm reserves was evaluated.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offsprings: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

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

Postmortem examinations (parental animals):

SACRIFICE
After the end of the experiment, the animals were euthanized by intraperitoneal administration of Morbital at a dose of 200 mg/kg b.w. and subjected to the gross examination.
All adult animals from groups 0, 1, 2, and 3 were sacrificed on the last day of experiment, i.e.:
- males – after pre-mating, mating, and post-mating periods (28 days),
- females – after pre-mating, mating, gestation, and 4-day lactation periods (40 - 47 days).

GROSS NECROPSY
All animals were subjected to the detailed gross examination involving the observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities and all organs with their content. In case of adult females, the numbers of implantation sites and corpora lutea were determined.

HISTOPATHOLOGY / ORGAN WEIGHTS
After the experiment, both absolute and relative weights of the testicles, epididymides, and ovaries of all adult animals were determined [SOP/P/10].
Relative weights of internal organs were calculated on the basis of absolute weights. Body weights of living animals were a point of reference.
The following organs and tissues were collected:
- testicles, epididymides, prostate, and vesicular and coagulating glands of all parental males,
- ovaries of all parental females,
- liver of the one male from group 2 in which the macroscopic change in the right lobe was stated.
The testicles and the epididymides were fixed in modified Davidson's fluid. The remaining organs listed above were fixed in a 10% solution of formalin. Preserved samples of organs were embedded in paraffin, and histopathological slides were stained using hematoxylin and eosin. The slides prepared in this way were evaluated under a light microscope (100x, 200x, and 400x).
The aim of the routine histopathological examination of collected organs and tissues of parental animals was to determine potential circulatory disorders, and inflammatory, reverse, or progressive changes. In addition to that, detailed examinations of the testicles and epididymides (males) and ovaries (females) were conducted to determine potential disorders of spermatogenesis and oogenesis. In case of the testicles, spermatogenetic cells (spermatogonia, spermatocytes, and spermatids) were examined. The examination involved the evaluation of the conformation of individual layers of the genital epithelium and the seminiferous tubule lumen content to exclude the presence of immature forms of spermatogenetic cells or giant cells. For the epididymides, the presence of sperm reserves was evaluated. In case of the ovaries, the follicles structure at different stages of their development and the structure of corpora lutea were assessed.

Postmortem examinations (offspring):

Dead pups (with the exception of pups eaten by their mothers) and the ones euthanized by intraperitoneal administration of Morbital at a dose of 200 mg/kg b.w. were examined for gross abnormalities on day 4 post-partum. The examinations involved observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content.

Statistics:

The results (average values and standard deviation) are presented in tables.
The treated groups, i.e. groups 1, 2, and 3 were compared to the control group.
The body weight results were statistically analyzed using Dunnett’s test (p ≤ 0.05).
Food intake is summarized in tables. The amount of data obtained in the study was insufficient because there were only 4 cages in each group and there were three animals housed in each cage. Hence, no statistical analyses were performed. Only food intake by females (gestation and lactation periods) was statistically analyzed.
Absolute and relative weights of internal organs as well as the numbers of implantation sites and corpora lutea were statistically evaluated using Dunnett’s test (p≤0.05).
The statistical analyses were conducted using Microsoft Excel 2002 and 2007, and Statistica 10 Pl.

Reproductive indices:

The following indices were counted for each experimental group:
Fertility of parental animals:
- Mating index for males:
the number of males showing evidence of copulation/the total number of mated males x 100
- Mating index for females:
the number of sperm-positive females/the total number of mated females x 100
- Fertility index for males:
the number of males that impregnated females/the total number of mated males x 100
- Fertility index for females:
the number of pregnant females/the total number of sperm-positive females x 100
- Pregnancy index:
the number of litters with live births/the number of pregnant females x 100.

Offspring viability indices:

The following indices were counted for each experimental group:
Offspring survival:
- Index for live births:
the total number of live newborns/the total number of newborns x 100
- Index for 4-day survival:
the number of live pups after 4 days/the number of live newborns x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

Mortality and morbidity of adult animals were not observed. During the entire experiment, there were no differences in appearance and behaviour of parental animals between the treated and the control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During the experiment there were no statistically significant differences in average body weights of males and females between the treated and the control groups. No differences in food intake were observed, either.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the experiment there were no statistically significant differences in average body weights of males and females between the treated and the control groups. No differences in food intake were observed, either.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No substance- or dose-dependent increases in histopathological lesions were observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

Mortality and morbidity of adult animals were not observed. All parental animals survived the experiment.

During the entire experiment, there were no differences in appearance and behaviour of parental animals between the treated and the control groups. However, alopecia was observed in two females from group 2 and two females from group 3.
Alopecia on the abdomen was observed in one female (no. 17) from group 2 and one female (no. 20) from group 3. Alopecia on the forelimbs was observed in two females (no. 17 and 23) from group 2 and one female (no. 21) from group 3 (transiently). Alopecia on the hindlimbs was observed in one female (no. 17) from group 2.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

During the experiment (pre-mating and after-mating periods), there were no statistically significant differences in average body weights of males between the treated and the control groups.
During the pre-mating, the gestation and the lactation periods, there were no statistically significant differences in average body weights of females between the treated and the control groups.

During the pre-mating period food intake by males and females from the treated and the control groups was similar. No statistical analyses of food intake were conducted, because the amount of data was insufficient (there were only 4 cages in each group and there were three animals housed in each cage). Therefore, it was impossible to clearly determine the relationship between the treated and the control groups.
During the gestation period, there were no statistically significant differences in food intake by females between the treated and the control groups.
During the lactation period, there were no statistically significant differences in food intake by females between the treated and the control groups.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Twelve females were mated with twelve males from the control group and groups 1, 2, and 3 during the scheduled period of mating. The average numbers of days needed for copulation (the day when males and females were housed together was day 0) were 2.3 in the control group, 2.5 in group 1, 2.8 in group 2, and 2.0 in group 3.
Twelve females from the control group and groups 1, 2, and 3 were sperm-positive. One female in group 2 was not pregnant. The remaining females were pregnant. Twelve females from the control group and groups 1 and 3, and eleven females from group 2 delivered offspring.
The mating indices for males and females in the control and the treated groups were 100.
The fertility indices for males and females in group 2 were lower, i.e. 91.7. The fertility indices in the control group and groups 1 and 3 were 100. The pregnancy indices in the control group and groups 1, 2, and 3 were 100.0.
The length of gestation in the treated and the control groups was similar, i.e. 22.2 days in the control group, 22.3 days in groups 1 and 3, and 22.1 in group 2.

Twelve females from the control group delivered live births. The average number of pups in a litter was 13.7, i.e. 57.3% were males and 42.7% were females. The number of pups in a litter ranged from 11 to 16. The number of live births in litter was 13.7.
Twelve females from group 1 delivered live births. The average number of pups in a litter was 13.2, i.e. 46.8% were males and 53.2% were females. The number of pups in a litter ranged from 8 to 16. The number of live births in litter was 13.2.
Eleven females from group 2 delivered live births. The average number of pups in a litter was 14.4, i.e. 51.3% were males and 48.7% were females. The number of pups in a litter ranged from 11 to 16. The number of live births in litter was 14.4.
Twelve females from group 3 delivered live births. The average number of pups in a litter was 11.9, i.e. 46.2% were males and 53.8% were females. The number of pups in a litter ranged from 8 to 15. The number of live births in litter was 11.8.


ORGAN WEIGHTS (PARENTAL ANIMALS)

The analysis of absolute and relative weights of internal organs of parental animals from groups 0, 1, 2, and 3 showed a statistically significant change, i.e. an increase in absolute weights of the testicles in males from group 1.
There were no statistically significant changes in absolute and relative weights of the remaining organs in any other group.


GROSS PATHOLOGY (PARENTAL ANIMALS)

There were no gross changes in groups 0, 1, and 3. As for group 2, an inflammatory focus with a diameter about of 0.5 cm in the right liver lobe and hyperemia of the liver in 1 male rat (no. 10) were observed.
There were no statistically significant changes in the numbers of corpora lutea and implantation sites.


HISTOPATHOLOGY (PARENTAL ANIMALS)

The following changes were observed:
group 0:
prostate
- lymphocytic infiltrations in 3 males
group 1:
prostate
- lymphocytic infiltrations in 4 males
group 2:
prostate
- lymphocytic infiltrations in 3 males
liver
- focal lymphocytic infiltration in the right lobe in 1 male
- hyperemia in 1 male
group 3:
prostate
- lymphocytic infiltrations in 2 males.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects; no adverse effects were noted at the highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

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

Description (incidence and severity):

The macroscopic examination of the dead pups and the ones euthanized on day 4 post-partum did not reveal any pathological changes or malformations

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)

There were 623 pups. One of them was born dead (1 male from group 3), whereas one pup died on the first day of life (1 female from group 2). Four pups (1 male and 3 females) from the control group, one pup (1 male) from group 1, one pup (1 male) from group 2, and two pups (2 females) from group 3 did not survive up to the 4th day of life. They were eaten by their mothers.

Four pups (1 male and 3 females) from the control group, one pup (1 male) from group 1, two pups (1 male and 1 female) from group 2, and two pups (2 females) from group 3 did not survive up to the 4th day of life.
The index of live births was 100 in control group and group 1 and 2, and 99.3 in group 3. The survival indices up to the 4th day of life were 97.6 in the control group, 99.4 in group 1, 98.7 in group 2, and 98.6 in group 3. The percentages of pup mortality were 2.4% in the control group, 0.6% in group 1, 1.3% in group 2, and 1.4% in group 3.


BODY WEIGHT (OFFSPRING)

There were no differences in bodyweigths on day 0 and 4 observed


GROSS PATHOLOGY (OFFSPRING)

The macroscopic examination of the dead pups and the ones euthanized on day 4 post-partum did not reveal any pathological changes or malformations. The eaten pups were not examined macroscopically.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects; no adverse effects were noted at the highest dose tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Due to space limitations, see attachment for tables.

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 421 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the toxicity to reproduction of 3-(cyclohexylamino)-propane sulfonic acid (screening study). The information gained from the screening study suffices with both regarding the tonnage band and safety assessment, as not any indication of adverse effects were observed up to the highest dose tested.

On the basis of the results of clinical observations, and body weight and food intake measurements, it may be concluded that the test item at the doses of 50, 200, and 800 mg/kg b.w. (males and females) had no adverse effects.
On the basis of the results of pup body weight measurements and the evaluation of reproduction, it may be concluded that the test item at the doses of 50, 200, and 800 mg/kg b.w. had no adverse effects. Also, the test item at the doses of 50, 200, and 800 mg/kg b.w. had not fetotoxic effects.
On the basis of the results of the post-mortem examinations of adults and their offspring, it may be concluded that the test item at the doses of 50, 200, and 800 mg/kg b.w. had no adverse effects.
On the basis of the results of the reproduction/developmental toxicity screening test of CAPS on rats, the no observed adverse effect level (NOAEL) of the test item was determined. It was 800 mg/kg b.w. The test item at this dose did not affect reproductive performance of adult males and females as well as the development of offspring up to 4th day of life.

In consequence, the test substance does not need to be regarded as reproductive or developmental toxicant.

Executive summary:

In a toxicity to reproduction screening study according to OECD guideline 421, 3-(cyclohexylamino)-propane sulfonic acid was administered daily by gavage in distilled water to each 12 Wistar rats (Cmdb:WI); outbred) per sex andper dose at dose levels of 0, 50, 200 and 800 mg/kg b.w. The following observations were made:

 

Clinical studies

All parental adults survived the experiment. A stillbirth (one pup) and pups mortality (nine pups) were observed. However, they were not test item-related effects.

There were no differences in physical appearance and behaviour between the treated and the control groups (parents and pups). A few pathological clinical signs were observed during the experiment. They were not test item-related effects.

There were no statistically significant differences in body weights between treated and control parents. In food consumption there were no differences too. No changes in reproduction were stated.

There were no differences in appearance and behaviour between treated and control pups.

The mating indices for males and females, the fertility indices for males and females, and the pregnancy indices in the treated and the control groups were similar. It suggests that the test item at the doses of 50, 200, and 800 mg/kg b.w did not influence on parental fertility.

The test item did not influence the total number of pups in a litter, the numbers of live births and stillbirths in a litter, and the percentages of males and females in a litter in groups 1, 2, and 3. It proves the lack of harmful influence of the test item at the test doses on fetuses.

The analysis of the indices related to offspring shows no negative influence of the test item on the number of live births and survival to 4 days of life.

On the basis of the results of the clinical observations, and body weight and food intake measurements, it may be concluded that the test item at the doses of 50, 200, and 800 mg/kg b.w. had no adverse effects.

On the basis of the results of pup body weight measurements and the evaluation of reproduction, it may be concluded that the test item at the doses of 50, 200, and 800 mg/kg b.w. had no adverse effects. Also, the test item at the doses of 50, 200, and 800 mg/kg b.w. had no fetotoxic effects.

 

Post-mortem examinations

Parental animals from groups 0, 1, 2, and 3 exhibited no macroscopic lesions. The only exception was 1 male from group 2 in which an inflammatory focus in the right liver lobe was noticed. It was collected and examined histopathologically. It was not a test item-related effect.

The macroscopic examination of offspring did not reveal any lesions or malformations in live born, stillborn, and dead pups.

On the basis of the statistical analysis of the numbers of corpora lutea and implantation sites, it can be concluded that the test item did not cause any changes in the numbers of corpora lutea and implantation sites when compared to the control group.

The analysis of absolute and relative weights of the testicles showed a statistically significant change (i.e. an increase in absolute weights in males in group 1). It was not a test item-related effect.

The analysis of absolute and relative weights of the epididymides and ovaries did not show any statistically significant changes.

The histopathological examination of collected organs and tissues of the parental animals revealed a few lesions in the prostate and liver (as for the liver, there was only one case). They were not test item-related effects.

On the basis of the results of the post-mortem examinations of the parental males and females as well as their offspring, it may be concluded that the test item at the doses of 50, 200, and 800 mg/kg b.w. had no adverse effects on the reproductive system of adults and the development of offspring.

 

Summary:

On the basis of the results of the reproduction/developmental toxicity screening test of (3-(cyclohexylamino)-propane sulfonic acid) on rats, the no observed adverse effect level (NOAEL) of the test item was determined. It was 800 mg/kg b.w. The test item at the doses of 50, 200, and 800 mg/kg b.w. had no toxic effects on reproductive performance of males and females as well as the development of offspring up to day 4 of life.