4-(cyclohexylamino)butane-1-sulfonic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Remarks:

Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 December 2019 - 22 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 December 2019 - 22 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test

Version / remarks:

2000

Principles of method if other than guideline:

In addition, the procedures described in this study plan essentially conform to the following guidelines:
• OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
• EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
• EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
• OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
• EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

other: Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks (males); 13-14 weeks (females)
- Weight at study initiation: 271 - 330 g (males); 201 - 247 g (females)
- Fasting period before study: no
- Housing:
Pretest (females only) and pre-mating period: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages
Mating phase: males and females were cohabitated on a 1:1 basis in Macrolon plastic cages(MIII type)
Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage ((Macrolon plastic cages, MIV type); females were individually housed in Macrolon plastic cages(MIII type).
Lactation phase: females were housed in Macrolon plastic cages(MIII type).

Pups were housed with the dam, except during locomotor activity monitoring of the dams.
The cages contained appropriate bedding and were equipped with water bottles. Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.

- Diet: ad libitum (except during designated procedures); Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum; Municipal tap water. Periodic analysis of the water was performed and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 21°C (actual range)
- Humidity: 44 to 55% (actual range)
- Air changes: Ten or greater air changes per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 11 Feb 2020 To: 17 April 2020

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

elix

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a clear solution (upon visual inspection), filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

Details on mating procedure:

- M/F ratio per cage:1:1 basis within the same treatment group
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis during week 1 of treatment using a validated analytical procedure. Concentration analysis was performed in all groups. The results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.
Homogeneity analysis was performed for groups 2 and 4. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20191868) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Duration of treatment / exposure:

Administered for 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 41-52 days.

Frequency of treatment:

Once daily

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of 4-(cyclohexylamino)butane-1-sulfonic acid in rats (Test Facility Reference No. 20191867), and in an attempt to produce graded responses to the test item
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION : Yes
- Time schedule for examinations:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight
- How many animals: 5/sex/group
- Parameters examined: White blood cells (WBC), Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW),,Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Large unstained cells (LUC) (absolute), Mean corpuscular haemoglobin, concentration (MCHC), Platelets, Red blood cells (RBC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room.
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight
- How many animals: 5/sex/group
- Parameters examined: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholestero, Total protein, Sodium, Albumin, Potassium,Total Bilirubin, Chloride, Bile Acids ,Calcium, Urea, Inorganic Phosphate (Inorg. Phos)

URINALYSIS: No

THYROID HORMONE ANALYSIS: Yes
Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m. On PND 4 at culling, blood was collected from two surplus pups per litter. On PND 14-16, separate blood samples were collected from two pups per litter.
Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
Parameters examined: Measurement of total T4 was conducted for F0-males and on female PND 14-16 pups. For the F0-generation assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males and no treatment related changes in thyroid weight were recorded.

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Males during Week 4 of treatment and the females during the last week of lactation (i.e. PND 6-13)
- How many animals: 5/sex/group
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity (1-hour)

Oestrous cyclicity (parental animals):

Time schedule: daily, for all females beginning 14 days prior to treatment and the first 14 days of treatment and during mating until evidence of copulation was observed
- Examination: examining the vaginal cytology of samples obtained by vaginal lavage

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight.
For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter 4/sex/litter (as nearly as possible); excess pups were euthanized.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
postnatal mortality, clinical signs (at least once daily), body weights (PND 1, 4, 7 and 13), sex (externally determined on PND 1 and 4), Anogenital distance (AGD), presence of nipples/areolae in male pups (PND 13).

THYROID HORMONE ANALYSIS
On PND 4 at culling, blood was collected from two surplus pups per litter. On PND 14-16, separate blood samples were collected from two pups per litter. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after a minimum of 28 days of administration
- Maternal animals: All surviving animals, PND 14-16 or after failure to deliver

GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS: Yes
- How many animals: Selected animals (5/sex/group) and non-selected (remaining) animals
- Organs weighed for all selected animals: brain, cervixa, epididymis, adrenal gland, coagulation gland, parathyroid gland, prostate gland, seminal vesicle gland, thyroid gland, heart
kidney, liver, Ovaries, spleen, testes, thymus, uterus
- Organs weighed for all remaining animals: epididymis, coagulation gland, parathyroid gland, prostate gland, seminal vesicle gland, thyroid gland, testes

HISTOPATHOLOGY: Yes
- Selected animals: bone marrow, femur, sternum, brain (eight levels), cervix, epididymidesa, eye, adrenal gland, coagulation gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, cecum, colon, rectum, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscles, sciatic nerve, ovaries, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testes, thymus, trachea, urinary bladder, uterus, vagina
- Males that failed to sire (except for males which were selected) and females that failed to deliver pups: cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Remaining animals: Gross lesions/masses.

Postmortem examinations (offspring):

SACRIFICE
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized. All remaining pups were euthanized on PND 14-16
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric:
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating index (%): (Number of females mated/Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females/Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 (after littering)) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in body weights and body weight gain were observed up to 1000 mg/kg bw/day. Any statistically significant changes in body weight gain (in males on several occasions in all dose groups) were considered to be unrelated to treatment with the test item, since no trend was apparent regarding dose level and duration of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item.
A statistically significant lower number of platelets in males treated at 100, 300 and 1000 mg/kg bw/day (0.87x, 0.87x and 0.78x of control, respectively) was considered not toxicologically relevant, since the mean value of the concurrent control group was relatively high (at the upper limit of the historical range) and means were within the range of historical control data .
Other statistically significant changes in hematology parameters (i.e. higher mean corpuscular haemoglobin (MCH) at 1000 mg/kg/bw/day (males), decreased haemoglobin at 100 and 300 mg/kg bw/day (females), decreased MCH at 100 mg/kg bw/day (females) and decreased mean corpuscular haemoglobin concentration (females)) were considered unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000 mg/kg bw/day.
Any statistically significant changes in clinical biochemistry parameters (i.e. increased sodium at 100 mg/kg/bw/day (males) and increased creatinine at 300 mg/kg bw/day (males), were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Thyroid hormone analyses:
Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg bw/day. Grip strength was similar between control and high dose animals. Motor activity assessment showed higher (although not statistically significant) mean values for total movements and ambulations in females at 300 and 1000 mg/kg bw/day at the end of the treatment period, particularly during test intervals 9, 10 and/or 11 (relative differences from control for sum of counts in all intervals: 1.29x and 1.21x for total movements and 1.38x and 1.32x for ambulations, respectively). These findings were not accompanied by clinical signs, no clear dose response was observed and the total movements and ambulations decreased to similar activity as concurrent control and historical control data during the final test interval. Therefore, the temporary increased motor activity in females at 300 and 1000 mg/kg bw/day was considered to be of no toxicological significance.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations. In the cecum of two females treated at 100 and 1000 mg/kg bw/day, minimal increased apoptosis in the villi were noted. In both control females and females treated at 300 mg/kg bw/day the finding was absent. Based on the absence of a dose-relationship, it is very unlikely that the finding is test item-related.
All histologic changes were considered to be incidental findings or within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
All females had regular cycles of 4 days during the pre-test period. During the treatment period, one female at 1000 mg/kg bw/day was acyclic. This female successfully mated within 3 days of pairing. Given the incidental nature and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation to treatment with the test item.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating index was considered not to be affected by treatment with the test item. The mating indices were 90, 100, 100 and 90% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Precoital time was considered not to be affected by treatment with the test item. All females showed evidence of mating within 4 days, except for one control Female.
Number of implantation sites was considered not to be affected by treatment with the test item. A relatively lower mean number of implantation sites was observed in the control group compared to the treated groups, which was attributed to one control female with two implantation sites. The fertility indices were 100, 100, 90, and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
A total of one female at 300 mg/kg bw/day and two females at 1000 mg/kg bw/day were not pregnant. Since these cases of non-pregnancy were within the normal range, showed no related histopathology changes in reproductive organs, and given the absence of any reproductive/developmental toxicity, this was considered not to be related to treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects up to and including the highest dose level tested.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects up to and including the highest dose level tested.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment with the test item.
The live birth indices were 100, 99, 100 and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One pup at 100 mg/kg bw/day was found dead at first litter check. No toxicological relevance was attributed to this dead pup, since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item.
Viability indices were 99, 100, 100, 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One pup of the control group and two pups at 1000 mg/kg bw/day were missing on PND 2 or 4 before culling. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A trend for a slightly lower pup body weight at 1000 mg/kg bw/day was noted, with a statistically significant lower pup body weight (females only) on PND 13 (7% lower, compared to concurrent controls; means remained within the range of historical control data).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Litter size was considered not affected by treatment with the test item.
Mean live litter sizes were 10.6, 11.1, 11.4 and 12.3 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. A relatively lower mean litter size was observed in the control group compared to the treated groups, which was related to the low number of implantation sites in one control female.

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects up to and including the highest doe levels tested.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

DOSE FORMULATION ANALYSES

Accuracy: The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 1. The maximum contribution to the Group 2 samples was 0.000070%, taking the dilution factor into account. This contribution was regarded negligible.

Homogeneity: The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Summary of the Dose Range Finder (DRF)

A dose range finder (Test Facility Reference No. 20191867) was conducted to select dose levels for the main study (Test Facility Study No. 20191866), and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. Dosing of the DRF was initiated on 06 Jan 2020. The in-life phase of the DRF was completed on16 Jan 2020.

Dose Formulations

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution.

Samples for dose formulation analysis were not collected during the dose range finder, as concentration, homogeneity, and stability analysis was not performed. However, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 6 hours after preparation of the formulation. Homogeneity and stability of the test itemunder test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20191868).

Test System

On 23 Dec 2019, female Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. Females were 13-14 weeks old and weighed between 226 and 249 g at initiation of dosing.

On arrival and following assignment to groups at random at the discretion of the biotechnician, animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). At study assignment, each animal was identified using earmark and tailmark. The actual daily mean temperature during the study period was 20°C with an actual daily mean relative humidity of 43 to 53%.

Experimental Design

Group No.	Test Item Identification	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Females	Animal Numbers
1	4-(cyclohexylamino)butane-1-sulfonic acid	500	5	100	3	1-3
2	4-(cyclohexylamino)butane-1-sulfonic acid	1000	5	200	3	4-6

The test item and vehicle were administered to the appropriate animals by once daily oral gavage for 10 consecutive days.

The dose levels were selected based on the results of an acute oral toxicity study in rats (LD₅₀< 2000 mg/kg body weight, Test Facility Study No. 20191856).

In-life Procedures

Mortality:	Twice daily throughout the study.
Clinical Observations:	At least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing (for exceptions, seeAppendix8).
Body Weights:	On Day 1 prior to dosing and on Days 5 and 10.
Food Consumption	Over Days 1-5 and 5-10.

Terminal Procedures

All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy). Animals were not deprived of food prior to necropsy.Terminal body weight, kidney and liver weight were determined at scheduled necropsy and all gross lesions were recorded. Gross lesions were not retained, no organs were fixed and histopathological examination was not performed.

RESULTS

No signs of toxicity were noted at any dose level.

Parameter	500 mg/kg/day	1000 mg/kg/day
Mortality	No mortality.	No mortality.
Clinical appearance	No findings.	No findings.
Body weight	Normal. 1/3 animals (Female No. 2) showed slight body weight loss on Day 10.	Normal.
Food consumption	Normal.	Normal.
Macroscopic examination	No abnormalities noted.	Accessory liver was noted for 1/3 animals (Female No. 5).
Organ weights	Liver and kidney weights considered to be normal.	Liver and kidney weights considered to be normal.

 

CONCLUSION

Based on the results of the dose range finder, selected dose levels for the Main study were 100, 300 and 1000 mg/kg bw/day.

Since no clinical signs were observed in the dose range finder, clinical observations were conducted and functional observations were started in the Main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

Conclusions:

Based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, performed according to OECD TG 422 and in accordance with GLP principles, the parental and reproduction NOAEL of 4-(cyclohexylamino)butane-1-sulfonic acid is considered to be at least 1000 mg/kg bw/day.

Executive summary:

A Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD TG 422 and in accordance with GLP principles. Male and female rats (10/dose) were exposed via gavage to 0 (control), 100, 300 and 1000 mg/kg bw/day. There were no unscheduled deaths.No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, hematology, clotting parameters and clinical biochemistry (including male T4 thyroid hormone levels), macroscopic examination, organ weights, and microscopic examination). At 1000 mg/kg bw/day, mean pup body weights were marginally lower on PND 13 (7% decreased in female pups compared to controls). This represented only a minor change and means remained well within the range of historical control data. Moreover, there were no adverse changes in other developmental parameters. As such, this minor variation in pup body weights was considered not to represent an adverse effect on pup development. No other treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental and reproduction NOAEL is considered to be at least 1000 mg/kg bw/day

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 December 2019 - 22 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Principles of method if other than guideline:

In addition, the procedures described in this study plan essentially conform to the following guidelines:
• OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
• EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
• EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
• OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
• EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

other: Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks (males); 13-14 weeks (females)
- Weight at study initiation: 271 - 330 g (males); 201 - 247 g (females)
- Fasting period before study: no
- Housing:
Pretest (females only) and pre-mating period: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages
Mating phase: males and females were cohabitated on a 1:1 basis in Macrolon plastic cages(MIII type)
Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage ((Macrolon plastic cages, MIV type); females were individually housed in Macrolon plastic cages(MIII type).
Lactation phase: females were housed in Macrolon plastic cages(MIII type).
Pups were housed with the dam, except during locomotor activity monitoring of the dams.
The cages contained appropriate bedding and were equipped with water bottles. Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.

- Diet: ad libitum (except during designated procedures); Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum; Municipal tap water. Periodic analysis of the water was performed and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 21°C (actual range)
- Humidity: 44 to 55% (actual range)
- Air changes: Ten or greater air changes per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 11 Feb 2020 To: 17 April 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

water

Remarks:

elix

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a clear solution (upon visual inspection), filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis during week 1 of treatment using a validated analytical procedure. Concentration analysis was performed in all groups. The results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration. Homogeneity analysis was performed for groups 2 and 4. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20191868) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study

Duration of treatment / exposure:

Administered for 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 41-52 days

Frequency of treatment:

Once daily

Dose / conc.:

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

Remarks:

group 2

Dose / conc.:

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

Remarks:

group 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of 4-(cyclohexylamino)butane-1-sulfonic acid in rats (Test Facility Reference No. 20191867), and in an attempt to produce graded responses to the test item
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION : Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight
- How many animals: 5/sex/group
- Parameters examined: White blood cells (WBC), Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW),,Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Large unstained cells (LUC) (absolute), Mean corpuscular haemoglobin, concentration (MCHC), Platelets, Red blood cells (RBC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m.,due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room.
- Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight
- How many animals: 5/sex/group
- Parameters examined: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholestero, Total protein, Sodium, Albumin, Potassium,Total Bilirubin, Chloride, Bile Acids ,Calcium, Urea, Inorganic Phosphate (Inorg. Phos)

URINALYSIS: No

THYROID HORMONE ANALYSIS: Yes
Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m.
Animals fasted: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
Parameters examined: Measurement of total T4 was conducted for F0-males. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males and no treatment related changes in thyroid weight were recorded.

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: males during Week 4 of treatment and the females during the last week of lactation (i.e. PND 6-13)
- How many animals: 5/sex/group
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity (1-hour)

ESTROUS CYCLE: Yes
- Time schedule: daily, for all females beginning 14 days prior to treatment and the first 14 days of treatment and during mating until evidence of copulation was observed
- Examination: examining the vaginal cytology of samples obtained by vaginal lavage

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.


ORGAN WEIGHTS: Yes
- How many animals: Selected animals (5/sex/group) and non-selected (remaining) animals
- Organs weighed for all selected animals: brain, cervix, epididymis, adrenal gland, coagulation gland, parathyroid gland, prostate gland, seminal vesicle gland, thyroid gland, heart
kidney, liver, ovaries, spleen, testes, thymus, uterus
- Organs weighed for all remaining animals: epididymis, coagulation gland, parathyroid gland, prostate gland, seminal vesicle gland, thyroid gland, testes

HISTOPATHOLOGY: Yes
- Selected animals: bone marrow, femur, sternum, brain (eight levels), cervix, epididymidesa, eye, adrenal gland, coagulation gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, cecum, colon, rectum, liver, Lung, lymph node (mandibular and mesenteric site), skeletal muscles, sciatic nerve, ovaries, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testes, thymus, trachea, urinary bladder, uterus, vagina
- Males that failed to sire (except for males which were selected) and females that failed to deliver pups: cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Remaining animals: Gross lesions/masses.

For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric:
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in body weights and body weight gain were observed up to 1000 mg/kg bw/day. Any statistically significant changes in body weight gain (in males on several occasions in all dose groups) were considered to be unrelated to treatment with the test item, since no trend was apparent regarding dose level and duration of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item.
A statistically significant lower number of platelets in males treated at 100, 300 and 1000 mg/kg bw/day (0.87x, 0.87x and 0.78x of control, respectively) was considered not toxicologically relevant, since the mean value of the concurrent control group was relatively high (at the upper limit of the historical range) and means were within the range of historical control data .
Other statistically significant changes in hematology parameters (i.e. higher mean corpuscular haemoglobin (MCH) at 1000 mg/kg bw/day (males), decreased haemoglobin at 100 and 300 mg/kg bw/day (females), decreased MCH at 100 mg/kg bw/day (females) and decreased mean corpuscular haemoglobin concentration (females)) were considered unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000mg/kg bw/day.
Any statistically significant changes in clinical biochemistry parameters (i.e. increased sodium at 100 mg/kg bw/day (males) and increased creatinine at 300 mg/kg bw/day (males), were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Thyroid hormone analyses:
Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg bw/day.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg bw/day. Grip strength was similar between control and high dose animals.
Motor activity assessment showed higher (although not statistically significant) mean values for total movements and ambulations in females at 300 and 1000 mmg/kg bw/day at the end of the treatment period, particularly during test intervals 9, 10 and/or 11 (relative differences from control for sum of counts in all intervals: 1.29x and 1.21x for total movements and 1.38x and 1.32x for ambulations, respectively). These findings were not accompanied by clinical signs, no clear dose response was observed and the total movements and ambulations decreased to similar activity as concurrent control and historical control data during the final test interval. Therefore, the temporary increased motor activity in females at 300 and 1000 mg/kg bw/day was considered to be of no toxicological significance.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations. In the cecum of two females treated at 100 and 1000 mg/kg bw/day, minimal increased apoptosis in the villi were noted. In both control females and females treated at 300 mg/kg bw/day the finding was absent. Based on the absence of a dose-relationship, it is very unlikely that the finding is test item-related.
All histologic changes were considered to be incidental findings or within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
All females had regular cycles of 4 days during the pre-test period. During the treatment period, one female at 1000 mg/kg bw/day was acyclic. This female successfully mated within 3 days of pairing. Given the incidental nature and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation to treatment with the test item.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of adverse effects up to and including the highest dose level tested.

Key result

Critical effects observed:

no

HISTORICAL CONTROL DATA

Historical control data for Wistar Han rats; F₀-females(period 2015-2019)

Total movements: mean = 3356; P5 – P95 =1602-5613 (n=546)

Ambulations: mean = 811; P5 – P95 =546-331 (n=1428)

Historical control data for Wistar Han rats; F₀-males(period 2017-2019):

Platelets (10E9/L): mean = 726; P5 – P95 = 581-882 (n=330).

DOSE FORMULATION ANALYSES

Accuracy: The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 1. The maximum contribution to the Group 2 samples was 0.000070%, taking the dilution factor into account. This contribution was regarded negligible.

Homogeneity: The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Summary of the Dose Range Finder (DRF)

A dose range finder (Test Facility Reference No. 20191867) was conducted to select dose levels for the main study (Test Facility Study No. 20191866), and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. Dosing of the DRF was initiated on 06 Jan 2020. The in-life phase of the DRF was completed on16 Jan 2020.

Dose Formulations

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution.

Samples for dose formulation analysis were not collected during the dose range finder, as concentration, homogeneity, and stability analysis was not performed. However, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 6 hours after preparation of the formulation. Homogeneity and stability of the test itemunder test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20191868).

Test System

On 23 Dec 2019, female Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. Females were 13-14 weeks old and weighed between 226 and 249 g at initiation of dosing.

On arrival and following assignment to groups at random at the discretion of the biotechnician, animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). At study assignment, each animal was identified using earmark and tailmark. The actual daily mean temperature during the study period was 20°C with an actual daily mean relative humidity of 43 to 53%.

Experimental Design

Group No.	Test Item Identification	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Females	Animal Numbers
1	4-(cyclohexylamino)butane-1-sulfonic acid	500	5	100	3	1-3
2	4-(cyclohexylamino)butane-1-sulfonic acid	1000	5	200	3	4-6

The test item and vehicle were administered to the appropriate animals by once daily oral gavage for 10 consecutive days.

The dose levels were selected based on the results of an acute oral toxicity study in rats (LD₅₀< 2000 mg/kg body weight, Test Facility Study No. 20191856).

In-life Procedures

Mortality:	Twice daily throughout the study.
Clinical Observations:	At least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing (for exceptions, seeAppendix8).
Body Weights:	On Day 1 prior to dosing and on Days 5 and 10.
Food Consumption	Over Days 1-5 and 5-10.

Terminal Procedures

All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy). Animals were not deprived of food prior to necropsy.Terminal body weight, kidney and liver weight were determined at scheduled necropsy and all gross lesions were recorded. Gross lesions were not retained, no organs were fixed and histopathological examination was not performed.

RESULTS

No signs of toxicity were noted at any dose level.

Parameter	500 mg/kg/day	1000 mg/kg/day
Mortality	No mortality.	No mortality.
Clinical appearance	No findings.	No findings.
Body weight	Normal. 1/3 animals (Female No. 2) showed slight body weight loss on Day 10.	Normal.
Food consumption	Normal.	Normal.
Macroscopic examination	No abnormalities noted.	Accessory liver was noted for 1/3 animals (Female No. 5).
Organ weights	Liver and kidney weights considered to be normal.	Liver and kidney weights considered to be normal.

 

CONCLUSION

Based on the results of the dose range finder, selected dose levels for the Main study were 100, 300 and 1000 mg/kg bw/day.

Since no clinical signs were observed in the dose range finder, clinical observations were conducted and functional observations were started in the Main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

Conclusions:

Based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, performed according to OECD TG 422 and in accordance with GLP principles, the NOAEL of 4-(cyclohexylamino)butane-1-sulfonic acid is considered to be at least 1000 mg/kg bw/day

Executive summary:

A Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD TG 422 and in accordance with GLP principles. Male and female rats (10/dose) were exposed via gavage to 0 (control), 100, 300 and 1000 mg/kg bw/day. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. There were no unscheduled deaths. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, hematology, clotting parameters and clinical biochemistry (including male T4 thyroid hormone levels), macroscopic examination, organ weights, and microscopic examination). In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the Parental NOAEL is considered to be at least 1000 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test

Version / remarks:

2000

Principles of method if other than guideline:

In addition, the procedures described in this study plan essentially conform to the following guidelines:
• OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
• EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
• EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
• OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
• EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-(cyclohexylamino)butane-1-sulfonic acid

EC Number:

629-080-0

Cas Number:

161308-34-5

Molecular formula:

C10 H21 N O3 S

IUPAC Name:

4-(cyclohexylamino)butane-1-sulfonic acid

Test material form:

solid

Details on test material:

Physical Description: White solid
Storage Conditions: At room temperature

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage condition of test material: At room temperature
Stability in water:
- Stability for at least 24 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1 to 200 mg/mL (solutions)
- Stability for at least 8 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL (solutions)
- Stability of 0.5 mL samples for at least 3 weeks in the freezer (≤ -15°C) is confirmed over the concentration range 1 to 200 mg/mL (solutions)
Test item handling: No specific handling conditions required

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Treatment of test material prior to testing: none

Test animals

Species:

rat

Strain:

other: Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-11 weeks (males); 13-14 weeks (females)
- Weight at study initiation: 271 - 330 g (males); 201 - 247 g (females)
- Fasting period before study: no
- Housing:
Pretest (females only) and pre-mating period: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages
Mating phase: males and females were cohabitated on a 1:1 basis in Macrolon plastic cages(MIII type)
Post-mating phase: males were housed in their home cage with a maximum of 5 males/cage ((Macrolon plastic cages, MIV type); females were individually housed in Macrolon plastic cages(MIII type).
Lactation phase: females were housed in Macrolon plastic cages(MIII type).

Pups were housed with the dam, except during locomotor activity monitoring of the dams.
The cages contained appropriate bedding and were equipped with water bottles. Animals were socially housed for psychological/environmental enrichment and were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.

- Diet: ad libitum (except during designated procedures); Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum; Municipal tap water. Periodic analysis of the water was performed and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: 6 days



ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 21°C (actual range)
- Humidity: 44 to 55% (actual range)
- Air changes: Ten or greater air changes per hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 11 Feb 2020 To: 17 April 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

elix

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a clear solution (upon visual inspection), filled out in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis during week 1 of treatment using a validated analytical procedure. Concentration analysis was performed in all groups. The results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration. Homogeneity analysis was performed for groups 2 and 4. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% of target concentration.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20191868) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study

Details on mating procedure:

- M/F ratio per cage:1:1 basis within the same treatment group
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged: individually

Duration of treatment / exposure:

Administered for 7 days a week for a minimum of 28 days. Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-64 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 41-52 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

group 2

Doses / concentrations 2

Dose / conc.:

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

Remarks:

group 3

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of 4-(cyclohexylamino)butane-1-sulfonic acid in rats (Test Facility Reference No. 20191867), and in an attempt to produce graded responses to the test item
- Fasting period before blood sampling for clinical biochemistry: F0-females were not fasted overnight.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION : Yes
- Time schedule for examinations:
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: F0-females were not fasted overnight
- How many animals: 5/sex/group
- Parameters examined: White blood cells (WBC), Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW),,Neutrophils (absolute), Haemoglobin, Lymphocytes (absolute), Haematocrit, Monocytes (absolute), Mean corpuscular volume (MCV), Eosinophils (absolute), Mean corpuscular haemoglobin (MCH), Basophils (absolute), Large unstained cells (LUC) (absolute), Mean corpuscular haemoglobin, concentration (MCHC), Platelets, Red blood cells (RBC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room.
- Animals fasted: F0-females were not fasted overnight
- How many animals: 5/sex/group
- Parameters examined: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholestero, Total protein, Sodium, Albumin, Potassium,Total Bilirubin, Chloride, Bile Acids ,Calcium, Urea, Inorganic Phosphate (Inorg. Phos)

URINALYSIS: No

THYROID HORMONE ANALYSIS: Yes
Time schedule for collection of blood: Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m.
Animals fasted: Parameters examined:For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males and no treatment related changes in thyroid weight were recorded.

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: females during the last week of lactation (i.e. PND 6-13)
- How many animals: 5/sex/group
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity (1-hour)

ESTROUS CYCLE: Yes
- Time schedule: daily, for all females beginning 14 days prior to treatment and the first 14 days of treatment and during mating until evidence of copulation was observed
- Examination: examining the vaginal cytology of samples obtained by vaginal lavage

SACRIFICE
- Maternal animals: All surviving animals, PND 14-16 or after failure to deliver

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS: Yes
- How many animals: Selected animals (5/sex/group) and non-selected (remaining) animals
- Organs weighed for all selected animals: brain, cervix, adrenal gland, parathyroid gland, thyroid gland, heart, kidney, liver, ovaries, spleen, thymus, uterus
- Organs weighed for all remaining animals: parathyroid gland, thyroid gland

HISTOPATHOLOGY: Yes
- Selected animals: bone marrow, femur, sternum, brain (eight levels), cervix, eye, adrenal gland, mammary gland, parathyroidc gland, pituitary gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, cecum, colon, rectum, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscles, sciatic nerve, ovaries, duodenum, ileum, jejunum, spinal cord, spleen, stomach, thymus, trachea, urinary bladder, uterus, vagina
- Females that failed to deliver pups: cervix, ovaries, uterus and vagina.
- Remaining animals: Gross lesions/masses

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter 4/sex/litter (as nearly as possible); excess pups were euthanized

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
postnatal mortality, clinical signs (at least once daily), body weights (PND 1, 4, 7 and 13), sex (externally determined on PND 1 and 4), Anogenital distance (AGD), presence of nipples/areolae in male pups (PND 13).

THYROID HORMONE ANALYSIS
On PND 4 at culling, blood was collected from two surplus pups per litter. On PND 14-16, separate blood samples were collected from two pups per litter. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric:
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.

Non-Parametric:
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).

Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Indices:

Mating index (%): (Number of females mated/Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females/Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 (after littering)) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item.
Statistically significant changes in hematology parameters (decreased haemoglobin at 100 and 300 mg/kg bw/day (females), decreased MCH at 100 mg/kg/day (females) and decreased mean corpuscular haemoglobin concentration (females)) were considered unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000 mg/kg bw/day.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals up to 1000 mg/kg bw/day. Grip strength was similar between control and high dose animals.
Motor activity assessment showed higher (although not statistically significant) mean values for total movements and ambulations in females at 300 and 1000 mg/kg bw/day at the end of the treatment period, particularly during test intervals 9, 10 and/or 11 (relative differences from control for sum of counts in all intervals: 1.29x and 1.21x for total movements and 1.38x and 1.32x for ambulations, respectively). These findings were not accompanied by clinical signs, no clear dose response was observed and the total movements and ambulations decreased to similar activity as concurrent control and historical control data during the final test interval. Therefore, the temporary increased motor activity in females at 300 and 1000 mg/kg bw/day was considered to be of no toxicological significance.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations. In the cecum of two females treated at 100 and 1000 mg/kg bw/day, minimal increased apoptosis in the villi were noted. In both control females and females treated at 300 mg/kg bw/day the finding was absent. Based on the absence of a dose-relationship, it is very unlikely that the finding is test item-related.
All histologic changes were considered to be incidental findings or within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
All females had regular cycles of 4 days during the pre-test period. During the treatment period, one female at 1000 mg/kg bw/day was acyclic. This female successfully mated within 3 days of pairing. Given the incidental nature and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation to treatment with the test item. No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 90, 93, 89 and 91% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were considered not to be affected by treatment with the test item.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The gestation index was 100% for all groups.

Other effects:

no effects observed

Description (incidence and severity):

No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: absence of adverse effects up to and including the highest dose level tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment with the test item.
The statistical significant changes in sex ratio observed at 300 and 1000 mg/kg bw/day were considered not to be toxicologically relevant, since a relatively high number of males was noted in the control group.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was considered not affected by treatment with the test item.
Mean live litter sizes were 10.6, 11.1, 11.4 and 12.3 living pups/litter for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. A relatively lower mean litter size was observed in the control group compared to the treated groups, which was related to the low number of implantation sites in one control female. A trend for a slightly lower pup body weight at 1000 mg/kg was noted, with a statistically significant lower pup body weight (females only) on PND 13 (7% lower, compared to concurrent controls; means remained within the range of historical control data).

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item.
Viability indices were 99, 100, 100, 98% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
One pup of the control group and two pups at 1000 mg/kg bw/day were missing on PND 2 or 4 before culling. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for all groups.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects up to and including the highest dose level tested

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

DOSE FORMULATION ANALYSES

Accuracy: The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Week 1. The maximum contribution to the Group 2 samples was 0.000070%, taking the dilution factor into account. This contribution was regarded negligible.

Homogeneity: The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Summary of the Dose Range Finder (DRF)

A dose range finder (Test Facility Reference No. 20191867) was conducted to select dose levels for the main study (Test Facility Study No. 20191866), and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. Dosing of the DRF was initiated on 06 Jan 2020. The in-life phase of the DRF was completed on16 Jan 2020.

Dose Formulations

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution.

Samples for dose formulation analysis were not collected during the dose range finder, as concentration, homogeneity, and stability analysis was not performed. However, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 6 hours after preparation of the formulation. Homogeneity and stability of the test itemunder test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20191868).

Test System

On 23 Dec 2019, female Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. Females were 13-14 weeks old and weighed between 226 and 249 g at initiation of dosing.

On arrival and following assignment to groups at random at the discretion of the biotechnician, animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). At study assignment, each animal was identified using earmark and tailmark. The actual daily mean temperature during the study period was 20°C with an actual daily mean relative humidity of 43 to 53%.

Experimental Design

Group No.	Test Item Identification	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Females	Animal Numbers
1	4-(cyclohexylamino)butane-1-sulfonic acid	500	5	100	3	1-3
2	4-(cyclohexylamino)butane-1-sulfonic acid	1000	5	200	3	4-6

The test item and vehicle were administered to the appropriate animals by once daily oral gavage for 10 consecutive days.

The dose levels were selected based on the results of an acute oral toxicity study in rats (LD₅₀< 2000 mg/kg body weight, Test Facility Study No. 20191856).

In-life Procedures

Mortality:	Twice daily throughout the study.
Clinical Observations:	At least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing (for exceptions, seeAppendix8).
Body Weights:	On Day 1 prior to dosing and on Days 5 and 10.
Food Consumption	Over Days 1-5 and 5-10.

Terminal Procedures

All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy). Animals were not deprived of food prior to necropsy.Terminal body weight, kidney and liver weight were determined at scheduled necropsy and all gross lesions were recorded. Gross lesions were not retained, no organs were fixed and histopathological examination was not performed.

RESULTS

No signs of toxicity were noted at any dose level.

Parameter	500 mg/kg/day	1000 mg/kg/day
Mortality	No mortality.	No mortality.
Clinical appearance	No findings.	No findings.
Body weight	Normal. 1/3 animals (Female No. 2) showed slight body weight loss on Day 10.	Normal.
Food consumption	Normal.	Normal.
Macroscopic examination	No abnormalities noted.	Accessory liver was noted for 1/3 animals (Female No. 5).
Organ weights	Liver and kidney weights considered to be normal.	Liver and kidney weights considered to be normal.

 

CONCLUSION

Based on the results of the dose range finder, selected dose levels for the Main study were 100, 300 and 1000 mg/kg bw/day.

Since no clinical signs were observed in the dose range finder, clinical observations were conducted and functional observations were started in the Main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

Applicant's summary and conclusion

Conclusions:

Based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, performed according to OECD TG 422 and in accordance with GLP principles, the maternal and developmental NOAEL of 4-(cyclohexylamino)butane-1-sulfonic acid is considered to be at least 1000 mg/kg bw/day

Executive summary:

A Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed according to OECD TG 422 and in accordance with GLP principles. Male and female rats (10/dose) were exposed via gavage to 0 (control), 100, 300 and 1000 mg/kg bw/day. There were no unscheduled deaths. No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, hematology, clotting parameters and clinical biochemistry (including male T4 thyroid hormone levels), macroscopic examination, organ weights, and microscopic examination). At 1000 mg/kg bw/day, mean pup body weights were marginally lower on PND 13 (7% decreased in female pups compared to controls). This represented only a minor change and means remained well within the range of historical control data. Moreover, there were no adverse changes in other developmental parameters. As such, this minor variation in pup body weights was considered not to represent an adverse effect on pup development. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the maternal and developmental NOAEL of 4-(cyclohexylamino)butane-1-sulfonic acid is considered to be at least 1000 mg/kg bw/day