Sulphamidic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD 443 study a daily administration of Sulfamic acid by oral gavage to Crl:WI (Wistar) rats at dose levels of 50, 150 and 500 mg/kg bw/day for a minimum of 17 weeks (Parental generation), followed by F1 generation pups selected for daily treatment by oral administration from weaning and terminated after the end of their treatment period (Cohort 1A), did not produce any macroscopic and microscopic test item-related findings.

There were no changes seen in the study that would suggest a generational effect of the test item on any of the evaluated parameters.

In conclusion, under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) values for Sulfamic acid was considered to be as follows:

• Based on test item effect on body weight, the NOAEL for systemic toxicity of parental generation: 500 mg/kg bw/day.


• Based on the effect on the reproductive parameters of parental animals (apparent lower male fertility at the High dose, and equivocal morphological sperm observations, restricted to the F0 generation), the NOAEL for reproductive toxicity of parental generation: 150 mg/kg bw/day.


• Due to lack of any test item effect on the body weight or food consumption of F1 generation, the NOAEL for physical development of F1 pups: 500 mg/kg bw/day.


• Due to lack of any test item effect on the sexual development of F1 generation, the NOAEL for sexual development of F1 pups: 500 mg/kg bw/day.


• No indication of any neurotoxic effect of the test item was indicated in the study.


• No indication of any immunotoxic effect of the test item was indicated in the study.


• No endocrine disruptor effect of the test item was indicated in the study.

As the effect on lower male fertility and sperm morphology observed in the P0 High dose group was low, but statistically significant, not dose dependent, and was also not seen in the Cohort 1A and Cohort 1B animals, this finding is not considered relevant for classification.

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019 - 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Dose range finding study (DRF)

Reason / purpose for cross-reference:

reference to other study

Remarks:

dose range finding study (DRF)

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

the study design (basic test design without extensions) was agreed before with the competent authority.

Specific details on test material used for the study:

Name: Sulfamic acid
Synonyms: Sulphamic acid, sulphamidic acid
Molecular formula: H3NO3S
Molecular weight: 97.08 g/mol
CAS no.: 5329-14-6
EC no.: 226-218-8
Description: White crystalline solid, odourless
Purity: 99.86% (as shown on the Certificate of Analysis)
Expiry date: 04 September 2020

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI (Wistar) rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl:WI (Wistar) rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
Hygienic level: Standard laboratory conditions
Justification of species/strain: The rat is regarded as suitable species for toxicology and reproduction studies. The Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Number of animals: Parental (P) generation: 96 male and 96 female rats (24 animals/sex/group, 4 groups). A sufficient number of spare animals (22/sex) was ordered; these animals were allocated to the spare colony of the Test Facility after the first month of the study had been finished.
Age of animals: Young adult rats, 7-8 weeks old at the initiation of study, and 17-18 weeks at mating. No siblings were used in the study.
Body weight range: Males: 310-375 g, females: 210-247 g at the start of the treatment; values did not exceed ±20% of the mean weight for each sex.
Acclimation period: 14 days
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room number: 525, 524
Cage type: Type II and/or III polycarbonate
Bedding: SAFE ¾-S certified wooden chips produced by J. Rettenmaier & Söhne GmbH + Co. KG were used in the study. Details of bedding quality are archived with the raw data.
Nesting: SAFE crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co. KG were available to animals during the study. Details of nest building material quality are archived with the raw data.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.7-26.1 ℃ (target range: 19-25 ℃)
Relative humidity: 24-74% (target range: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively. Males were caged individually after mating had been completed. Animals of F1 generation were group-housed (up to 3 animals of the same sex per cage) after the cohort selection, wherever possible. Group housing allowed social interaction, and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting). Nest building material was added to the cages. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels) produced by LBS (Serving Biotechnology) Ltd. were also provided to the animals for additional enrichment, details of the quality were documented in the raw data.
Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study. All the relevant documentation is kept and archived in the raw data.
Food and water supply: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – Breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, ad libitum. The description of the diet and the analytical certificate for the used batch were included in the raw data.
During the study animals received tap water from municipal supply, as for human consumption from 500 ml bottle, ad libitum. Water quality control analysis was performed once every three months, and microbiological assessment was performed monthly by the Veszprém County Institute of State Public Health and Medical Officer Service. The quality control results were included in the raw data.
Food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The dosing formulations were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, in each morning, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 10 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted to the current body weight of the animals. The control animals received the vehicle at the same administration volume daily in the same way.

Details on mating procedure:

Mating procedure
Mating began after the animals had attained full sexual maturity, after 10 weeks of treatment (pre-mating period). Mating was performed with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred. Mating pairs were clearly identified in the data.
For confirmation of mating, a vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Gestational Day 0 or GD 0, and was defined as start of pregnancy according to the relevant OECD No. 414 guideline). Sperm-positive females were caged individually.
Due to the high number of unsuccessful mating process, additional (second) mating for one control (#1501), five Low dose (#2506, #2509, #2514, #2518 and #2520) , three Mid dose (#3511, #3512 and #3519) and seven High dose (#4510, #4513, #4515, #4519, #4522, #4523 and #4524) females was applied from Day 106. No mating was performed for Cohort 1A or Cohort 1B animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item formulations were freshly prepared every day. The test item was dissolved in the selected vehicle to the appropriate concentrations according to the selected dose volume. The formulation was properly homogenized (by magnetic stirrer and/or vortex) before used for treatment. The formulations were visually assessed for apparent homogeneity (they were clear colourless solutions).
Analysis of test item formulations for concentration and homogeneity was performed using validated analytical method, (Test Facility Study code: 19/250-316AN). As specifically requested by the Sponsor, a titration method was selected for analysis of the study formulations. This was not a selective method, but the test item was a small molecule which was not volatile and has no special spectral absorbance, hence the generally used specific analytical methods of Gas Chromatography with flame ionization detector (GC-FID) or High Performance Liquid Chromatography with ultraviolet detection (HPLC-UV) could not be used. The only chemical added to the aquatic vehicle was the Sulfamic acid with a purity of 99.86%, so it was assumed that the measured acid was Sulfamic acid. The same titration method was used in the OECD No. 414 Prenatal Developmental Toxicity Study.
The pH of the formulations (During the treatment period, the pH of the different formulation was in the following ranges: 4.25-7.09 (Control), 1.29-1.70 (Low dose), 1.01-1.51 (Mid dose) and 0.70-1.21 (High dose) was checked before the first treatment and was recorded at each formulation analysis occasion except of one case. The selected analytical method did not verify chemical stability in the test system.
For formulation analysis top, middle and bottom duplicate samples (at least 5 mL each) were taken from test item formulations eight times during the study (during the first week and approximately equally spaced during the treatment period), one set to analyse (which can be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement on each occasion. Samples were analysed freshly. Additional samples were collected for other, confirmatory purpose.
The concentration of Sulfamic acid in the test item formulation samples was determined by measuring the acid reserve of the formulations (the amount of a NaOH solution (0.1M) which was necessary to cause the colour change of the pH indicator (phenolphthalein) in the defined volume test item formulation)( 0.1 mL 0.1 M NaOH was necessary to cause the colour change of the pH indicator in test item formulation containing 0.971 mg Sulfamic acid). Five titrations were performed from each sample of test item formulation or control.
Acceptance criteria of the concentration analysis was set according to the analytical method validation (100 ±10% of the nominal concentration).
Acceptance criteria of homogeneity was set according to the relevant SOP of the Test Facility and analytical method validation (the coefficient of variation (CV) of replicates had to be less than 10%).
Any sample not required for analysis was discarded following acceptance of the results by the Analyst and Study Director.
Raman spectroscopy of formulations
In order to gain more information about formulation preparation, additional analytical concentration measurements using Raman spectroscopy (applicable for structure identification) was performed twice during the study. Those analytical measurements were performed at a designated Test Site #2 under the control of a Principal Investigator #2 using a validated method.
For the additional measurements, samples were collected into clean glass containers twice during the study (10 mL formulations for the middle of the containers in duplicates; Set#1 and Set #2) and were immediately send at room temperature to the attention of the Principal Investigator. The measurements were carried out according to the analytical method validation study (Test Facility Study code: 19/250-901AN) under the control of the Principal Investigator using the Set #1 samples within 72 hours after the formulation preparation, while samples of Set #2 were back-up samples (used for confirmatory measurement on the first sampling occasion).
Acceptance criterion of the concentration analysis was set according to the analytical method validation (100 ± 10% of the nominal concentration). After the measurements had been finished, the Principal Investigator issued a Phase Report (a draft version was sent for review to the Study Director and Study Monitor). Any sample not required for analysis was discarded following acceptance of the results by the Principal Investigator #2 and Study Director.

The concentration and homogeneity of the test item in the dosing formulations was determined by using a validated titration method on eight occasions during the study. The summary is shown in Table 7.
Using the titration method, no test item was detected in the control samples at any occasion. The mean measured concentrations of test item formulations were in the range of 97-106% of their nominal concentrations on all occasions. These results were within the acceptable range (90-110% of the nominal concentration). Formulations were also proven to be homogenous. Detailed results of the formulation analysis are included in the Analytical Report (Appendix 6).
Test item formulations were considered to be adequately stable under the study conditions (formulation in the 2-60 mg/mL concentration range were shown in the validation study of the titration method to be stable for 24 hours when stored at room temperature and in the study they were used for treatment immediately after preparation; additional stability information by Raman spectroscopy are shown later in this section).
As the titration method was known to be non-specific, the concentrations of the test item formulation were additionally measured by a chemically specific method (Raman spectroscopy) on two occasions, summary is shown Table 8. No test item was detected in the control samples at any occasion.
At the first measurement, the Set #1 result of the Low dose formulation was lower than expected (87% of the nominal value). The corresponding sample of Set #2 was additionally measured, the result was at the low end of the acceptable range (90% of the nominal value).
At the second measurement, the mean recoveries for the samples were found to be 92%, 105% and 98% for the Low, Mid and High dose formulations, respectively. These values were within the acceptable range. Using this specific analytical method, test item formulations were proven to be stable at least for 3 days (High dose formulation) or 7 days (Low dose formulation) when stored at room temperature. Detailed results of this formulation analysis are included in the Phase Report of the Raman spectroscopy.
Overall, test item formulations were considered to be suitable for the study purposes.

Table 7: Summary of analytical results (titration)
Formulation occasion Nominal concentration
(mg/mL) Measured concentration
(mg/mL) Percentage of the nominal concentration
(%) RSD% (%)
31 October 2019 Controls 1-2 < 0.013 (< 1 drop) - -
5 5.11 ± 0.0556 102 1.04
15 15.1 ± 0.100 101 0.63
50 50.2 ± 0.105 100 0.20
07 November 2019 Controls 1-2 < 0.013 (< 1 drop) - -
5 5.08 ± 0.0258 102 0.48
15 15.1 ± 0.122 101 0.77
50 50.5 ± 0.83 101 0.16
08 January 2020 Controls 1-2 < 0.013 (< 1 drop) - -
5 5.18 ± 0.0223 104 0.41
15 15.5 ± 0.0526 103 0.32
50 50.3 ± 0.112 101 0.21
14 February 2020 Controls 1-2 < 0.013 (< 1 drop) - -
5 5.18 ± 0.0341 104 0.63
15 15.5 ± 0.0416 103 0.26
50 50.8 ± 0.105 102 0.20
30 March 2020 Controls 1-2 < 0.013 (< 1 drop) - -
5 5.12 ± 0.0515 102 0.96
15 15.7 ± 0.0911 105 0.55
50 50.4 ± 0.210 101 0.40
20 April 2020 Controls 1-2 < 0.013 (< 1 drop) - -
5 4.99 ± 0.0341 100 0.65
15 14.9 ± 0.0416 99 0.27
50 48.6 ± 0.214 97 0.42
07 May 2020 Controls 1-2 < 0.013 (< 1 drop) - -
5 5.03 ± 0.0210 101 0.40
15 14.8 ± 0.0766 99 0.49
50 48.5 ± 0.238 97 0.47
19 July 2020 Controls 1-2 < 0.013 (< 1 drop) - -
5 5.10 ± 0.0333 102 0.62
15 15.7 ± 0.1001 105 0.61
50 52.9 ± 0.170 106 0.31
Notes: Mean measured concentrations with 95% confidence interval are shown. Percentage of nominal concentration values are rounded. RSD: Relative Standard deviation.

Table 8: Summary of analytical results (Raman spectroscopy)
Formulation occasion Nominal concentration (mg/mL) Measured concentration (mg/mL) Percentage of the nominal concentration (%) RSD% (%)
26 March 2020 Control BQL - -
5§ 4.37 / 4.50 87 2.0
15 14.91 99 0.1
50 54.17 108 0.0
25 May 2020 Control BQL - -
4.96 4.56 92 1.6
15.03 15.75 105 0.3
50.98 50.25 98 1.4
Notes: Mean measured concentrations with 95% confidence interval are shown (mean results of the triplicate measurement of the same sample). Percentage of nominal concentration values are rounded. RSD: Relative Standard deviation. BQL: Below Quantification Limit.
§: Results of Set #1 / Set #2 are shown.

Duration of treatment / exposure:

Parental males (F0) were treated for 10 weeks pre-mating (this period fully covered the time required for epididymal transit of maturing spermatozoa and to allow the detection of post-testicular effects on sperm during the final stages of spermiogenesis and epididymal sperm maturation at mating), through the mating period and after that until they were no longer needed for assessment of reproductive effects (at least until weaning of the F1); a total of at least 17 weeks of treatment. All parental males were euthanised and subjected to necropsy examination.
Parental females (F0) were treated for 10 weeks pre-mating (this period fully covered several complete oestrous cycles and was sufficient to detect any adverse effects on cyclicity), and during the mating, gestation, and lactation periods until they were no longer needed for assessment of reproductive effects (after weaning of their litters), a total of at least 17 weeks of treatment. The day of birth (when parturition was complete) was defined as post-partum day 0 (PPD 0). Non-pregnant females or females not having delivered (#2518, #4513, #4524 and #2514) were maintained and sacrificed on Day 150.
F1 pups were treated for 69 days (Cohort 1A) or approximately 150 Days (Cohort 1B) started after weaning (on PND 21). Length of treatment was in line with the OECD No. 443 requirements.

Frequency of treatment:

The dosing formulations were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, in each morning, by oral gavage.

Dose / conc.:

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

Remarks:

low dose group

Dose / conc.:

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

Remarks:

mid dose group

Dose / conc.:

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

Remarks:

high dose group

No. of animals per sex per dose:

Twenty-four male and twenty-four female Wistar rats/group were treated in the parental generation via the oral route according to the experimental design.

Control animals:

yes, concurrent vehicle

Details on study design:

Planned changes to the Study Plan were made in the study on two occasions, those changes were documented by Study Plan amendments.

Due to technical reasons, temperature values (maximum of 26.1oC) and relative humidity values (minimum of 24% / maximum of 74%) outside the expected range of 19-25oC and 30-70% were recorded during the in-life phase. However, the observed minor differences in the environmental parameters observed for short periods (a total of 5 times, not more than 3 consecutive days in any of those cases) were considered not to adversely affect the results or integrity of the study as confirmed by the clinical veterinarian.

Due to technical oversight, the pH of the formulations was not checked on the 26 March 2020 sampling occasion. However, as results from all the other occasions exist, this fact was considered not to adversely affect the results or integrity of the study.

Formulation analysis by the titration method was performed on a total of eight occasions (although 6 was planned in the Study Plan) properly spaced during the treatment period, but no sampling was performed on the last week. As the required amount of information was collected, these facts were considered not to adversely affect the results or integrity of the study.

As for the applicable analytical method (titration) the source of water had extreme importance, additional titration step was performed for confirmation about the applicability of water from different sources, in order to have the available information in case of necessity (COVID-19 shortage).

Due to an unscheduled delay of data tabulation process, the Draft Report was issued later than indicated in the Study Plan. However, this deviation was considered to have no impact on the results or integrity of the study.

Parental animals: Observations and examinations:

Clinical observations
The principles and criteria summarised in the Humane Endpoints Guidance Document (OECD Guidance Document No. 19 on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation, 2000) were taken into consideration during clinical observation.
Adult animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day as practical. Animals showing severe clinical signs were sacrificed to prevent suffering, cannibalism or autolysis.
General clinical observations were made once a day (usually no general clinical observations were made on those days when detailed clinical observations were made). The animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable.
Detailed clinical observations were made on all adult animals at least prior to the first treatment (to allow for within-subject comparisons), at least weekly thereafter and on the day of necropsy. These observations were made outside the home cage in a standard arena, at similar times, generally during the morning. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
The sperm-positive parental females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat) on Gestation Day 13 and/or 14 (GD13 / GD14).
Furthermore, mated parental females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.
Body weight measurement
Parental (P) males were weighed with a precision of 1 g at randomisation (Day -8), on the first day of treatment (Day 1), then at least weekly through the pre-mating / mating periods and after that, and prior to necropsy.
Parental (P) females were weighed with the same precision at randomisation (Day -8), on the first day of treatment (Day 1), then at least weekly through the pre-mating / mating periods, on gestation days (GD) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22, and during lactation on post-partum days (PPD) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and prior to necropsy (or at death for animals found dead).
Food consumption measurement
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g at least once per week. Weekly food consumption was calculated for reporting purposes. Spillage (if any) was recorded and reported.
Food consumption of parental (P) males and females was measured at least weekly during the pre-mating period (on the same days as animal body weight measurements). Individual food consumption was not measured during cohabitation. Evaluation of food consumption of parental (P) males and females commenced again following the mating period until necropsy and was conducted at least weekly (on the same days as animal body weight measurements).
Water consumption measurement
As contradictory data were available, water consumption of parental males and females was evaluated with a precision of 1 mL in the first five weeks (from Days 1-34) during the pre-mating period by re-weighing the non-consumed water in the water bottles. Additional measurements were implemented during the gestation and lactation periods for female animals. Weekly water consumption was calculated for reporting purposes.
Neurological assessment
No neurological assessment was performed for parental (P) adult animals.
Ophthalmology evaluation
No ophthalmoscopic examination was conducted for parental (P) adult animals or for cohorts of the F1 generation.
Oestrous cycle monitoring
Parental (P) females were monitored daily for the stage of oestrous cycle by assessment of vaginal smears for two weeks before the start of mating, and during the mating period until evidence of mating was noted. The process of vaginal smear preparation is described in the mating procedure. The stage of oestrus was evaluated using these smears by competent personnel. Similarly, the oestrous cycle was monitored daily in Cohorts 1A and 1B females for two weeks after vaginal opening.
Observation of the delivery process and nursing instinct
Pregnant parental (P) females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible (all observations were recorded). Furthermore, any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy (Gestational Day 0, GD0) until the completion of parturition.
Dams were observed for signs of nest building with the bedding material and for covering their new-borns. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded. No such steps were performed in Cohort 1A or Cohort 1B animals.
CLINICAL PATHOLOGY
Blood sampling
All parental (P0) adult males and females were terminated when they were no longer needed for assessment of reproductive effects.
For terminal blood sampling, 10 males and 10 females/group were selected randomly (according to the general procedure of the Test Facility). All animals selected for blood sampling were fasted (overnight period of food deprivation; in case of females this step was performed after the litter had been culled).
Blood samples were collected sublingual (for thyroid hormone analysis) or by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. Four samples were taken from each animal: two tubes for haematology and thyroid hormone analysis (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL; 0.6-1.2 mL blood/tube), one for blood clotting times (in tubes with sodium citrate as anticoagulant; 1.2 mL blood/tube) and one to obtain serum (in tubes with no anticoagulant; 1.4 mL blood/tube) for clinical chemistry.
Urine sampling
Urine samples were collected overnight (for approximately 16 hours) with no access to food, but with access to water, in individual metabolism cages.
Haematology and blood clotting times
The parameters listed in Table 3 were evaluated in the selected parental animals. All the parameters measured for selected parental generation animals were also measured for all Cohort 1A and Cohort 1B animals.
Blood smears were prepared for all selected animals but not examined (as no test item-related effect was observed at the standard haematology evaluation). The smears were archived at the Test Facility.
Table 3: Summary of the evaluated haematology parameters
Parameter (Unit) Method
RBC Red Blood Cell (erythrocyte) count, (10¹²/L, M/µL) Automatic laser cell count
Hgb Haemoglobin Concentration (g/dL) Determination of cyan-methemoglobin absorbance
Hct Haematocrit (%) Calculated value
MCV Mean Corpuscular (erythrocyte) Volume (fL) Laser cell volume determination
MCH Mean Corpuscular (erythrocyte) Haemoglobin (pg) Calculated value
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration (g/dL) Calculated value
RDW Red Cell Distribution Width (erythrocyte) volume (%) Distribution Width Laser detection
Plt Platelet (thrombocyte) count (10⁹/L) K/µL Automatic laser cell count
MPV Mean Platelet (thrombocyte) volume (fL) Cell volume determination by laser
RETIC Reticulocyte (%) Comparative value based on laser light detection
WBC White Blood Cell (leukocyte) count, (10⁹/L) K/µL Automatic laser cell count
NE % Neutrophil (%) Cell differentiation based on myeloperoxidase activity
LY % Lymphocyte (%)
MO % Monocyte (%)
BA % Basophil (%)
EO % Eosinophil (%)
LUC % Large Unstained Cells (%)
RETIC % Reticulocyte (%)
NE Neutrophil (Abs.) K/µL Calculated value
LY Lymphocyte (Abs.) K/µL Calculated value
MO Monocyte (Abs.) K/µL Calculated value
BA Basophil (Abs.) K/µL Calculated value
EO Eosinophil (Abs.) K/µL Calculated value
LUC Large Unstained Cells (Abs.) K/µL Calculated value
RETIC Reticulocyte (Abs.) K/µL Calculated value

Table 4: Summary of the evaluated coagulation parameters
Parameter (Unit) Method
APTT Activated Partial Thromboplastin Time (sec) Factors of intrinsic coagulation system are activated by incubation the plasma with the optimal amount of phospholipids, a surface activator and calcium ions.
PT Prothrombin Time (sec) The coagulation cascade is activated by incubating plasma with the optimal amount of thromboplastin and calcium.
Fibrinogen (g/L) Clauss method

Clinical chemistry
The parameters shown in Table 5 were evaluated in the selected parental animals. All the parameters measured for selected parental generation animals were also measured for all Cohort 1A and Cohort 1B animals.
Table 5: Summary of the evaluated clinical chemistry parameters
Parameter (Unit) Method
Chol. - Cholesterol (mmol/L) Colorimetric test (540 nm)
Glucose - Blood sugar (mmol/L) Colorimetric test (540 nm)
Urea - Urea (mmol/L) Colorimetric test (670 nm)
Creat. - Creatinine (μmol/L) Two-point rate test (670 nm)
Na+ - Sodium (mmol/L) Potentiometric test
K+ - Potassium (mmol/L) Potentiometric test
Cl - Chloride (mmol/L) Potentiometric test
Ca++ - Calcium (mmol/L) Colorimetric test (680 nm)
Phos. - Phosphorus (mmol/L) Colorimetric test (680 nm)
T-BIL - Total Bilirubin (μmol/L) End-point colorimetric (dual-wavelength) test (400 & 460 nm)
AST/GOT - Aspartate Aminotransferase activity (U/L) Multiple-point rate test (340 nm)
ALT/GPT - Alanine Aminotransferase activity (U/L) Multiple-point rate test (340 nm)
GGT – Gamma Glutamyl Transferase activity (U/L) Multiple-point rate test (400 nm)
ALKP - Alkaline Phosphatase activity (U/L) Multiple-point rate test (400 nm)
Tot. prot. - Total Protein (g/L) Colorimetric test (540 nm)
Alb. - Albumin (g/L) Colorimetric test (630 nm)
A/G - Albumin/Globulin ratio Calculated value

Urinalysis
The parameters shown in Table 6 were evaluated in the selected parental animals using the urine collected from the metabolism cages. All the parameters measured for selected parental generation animals were also measured for all Cohort 1A and Cohort 1B animals.
Table 6: Summary of evaluated urinalysis parameters
Parameter Method
LEU / Leukocyte Reflection photometry
NIT / Nitrite Reflection photometry
pH Reflection photometry
PRO / Protein Reflection photometry
GLU / Glucose Reflection photometry
UBG / Urobilinogen Reflection photometry
BIL / Bilirubin Reflection photometry
KET / Ketones Reflection photometry
BLD/ERY / Blood/Erythrocytes Reflection photometry
SG / Specific Gravity Reflection photometry
SED / Sediment Microscopic examination
Volume Volumetric method
Colour, appearance, clarity Visual observation

Thyroid hormone analysis
For thyroid hormone analysis of selected parental (P) animals, blood samples (collected into tubes containing K3-EDTA as anticoagulant) were taken by sublingual sampling, and kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4 °C). The resulting plasma was divided in at least two aliquots (volume target was at least 125 µL for the first aliquot and at least 75 µL for the second aliquot if possible, remaining material - if any -was kept as third (back-up) aliquot) and stored in an ultra-freezer (-80 ±10 °C) until shipping for analysis.
Samples (Set #1) were shipped for thyroid hormone analysis on dry ice to the attention of the Principal Investigator #1 (PI #1) at Test Site #1 to the recipient and address shown here: Fabien Haule, Sample Manager, Charles River Laboratories Evreux, RN13 Route de Pacy, 27930 Miserey, France.
The thyroid hormone analysis was conducted using the validated methods under the control of the Principal Investigator #1 in compliance with the Test Site #1 relevant SOPs. Samples were assessed for T4 (125 µL aliquots) and TSH (75 µL aliquots) levels.
The method for T4 measurement was based on a solid phase extraction from plasma followed by an analysis of the extract by reverse phase liquid chromatography (HPLC) with MS/MS detection after ionization using an Electro Spray Interface (ESI, positive mode). All details of the analytical method were presented in the analytical procedure document which were retained in study raw data. The concentration range was 4.00 to 500 ng/mL for T4. The analytical procedure for the determination of T4 in rat plasma by LC MS/MS was developed at Test Site #1 (Charles River Laboratories Evreux). Complete validation was carried out prior to the analysis of the study samples. The validation results are summarized in separate validation report (Charles River Laboratories Evreux Study No. 39971 RDR).
The method for TSH measurement was based on the Luminex xMAP® technology, that is a bead-based immunoassay system in a microplate format. Microspheres coated with antibodies directed against TSH was color-coded with two different fluorescent dyes. After the TSH from rat plasma sample was captured by antibody-coupled beads, beads were incubated with a biotinylated-coupled antibody which was revealed using a Streptavidin-Phycoerythrin (SAPE) conjugate. The microsphere specific of TSH was identified by the emission wavelengths of the beads whereas the analyte in the tested sample was detected and quantified with SAPE. Each analyte concentration was then determined by interpolating Fluorescence Intensity (FI) values of the samples with a calibration curve in which the SAPE Fluorescence Intensity (FI) was plotted against the logarithm (base 10) of TSH concentrations. The analytical procedure for the determination of TSH in rat plasma by Luminex xMAP® technology was developed at Charles River Laboratories Evreux. Complete validation was carried out prior to the analysis of the study samples. The validation results are summarized in separate validation report (Charles River Laboratories Evreux Study No. 40045 RDR).
Assay of plasma samples
For T4 analysis, each batch consisted of:
- one reagent blank,
- blank samples (extracted BSA without analyte and Internal Standard),
- two 0 ng/mL standard samples (extracted BSA with the Internal Standard but without the analyte),
- methanol injections in order to avoid the any carry-over phenomenon,
- two calibration curves using seven calibration levels (4.00 - 500 ng/mL for T4) and injected at the beginning and at the end of the analytical sequence,
- QC samples (at least six) prepared in BSA at three concentration levels in duplicate (low-range, mid-range and near the upper limit),
- QC samples (n = 6) prepared in plasma at three concentration levels in duplicate (low-range, mid-range and near the upper limit),
- blank plasma (n = 3) spiked only with Internal Standards to determine the endogenous levels of T4,
- study samples.
For TSH analysis, each batch consisted of:
- a blank sample,
- a calibration curve with eight concentrations of calibration points (20.0 to 5000 pg/mL),
- six Quality Control (QC) samples: three concentration levels in duplicates, distributed at the beginning and at the end of the batch (120, 2500 and 3750 pg/mL),
- study samples.
The QC samples were regularly distributed over the batch. The QC samples were prepared using different standard solutions to those used for the calibration curve.
The results of the thyroid hormone analysis in the form of a Phase Report are included in the study report as an appendix.
Any sample not required for analysis will be discarded following acceptance of the results of the hormone analysis by the Principal Investigator #1 and Study Director (after the finalisation of the study report).
Part 2 animals
Blood sampling and sample shipment were made for thyroid hormone analysis (TSH, T4) of Cohort 1A selected animals in a similar way.
Other cohorts
Blood sampling for thyroid hormone analysis was made for all Cohort 1B animals (in order to provide sample if any additional data needed in case of equivocal results of Cohort 1A). However, samples were not analysed, as it was not deemed necessary based on the observed results.
Blood sampling was also made from surplus animals (not selected at litter standardisation on PND 4 or for cohort continuation on PND22) at termination. In those cases, one pooled sample was made for each litter using blood of two pups, where applicable. Shipment of those samples was made as described previously.

Oestrous cyclicity (parental animals):

see above

Sperm parameters (parental animals):

see above

Litter observations:

For F1 adult animals, body weight was recorded with a precision of 1 g at weaning (PND 21, prior to the start of treatment), then at least weekly, including the last treatment day and prior to necropsy (fasted at scheduled necropsy), or at death for animals found dead).
The determination of food consumption of Cohorts 1A and 1B of the F1 generation was also performed. The first measurement of given food was made at weaning (PND 21) The remaining, non-consumed food was weighed at least weekly with a precision of 1 g. Food hoppers were replenished more frequently if required.
Neurological assessment
In case of Cohort 1A animals, towards the end of the treatment period (in the period of Days 64-69) each animal of Cohort 1A was subjected to the functional observation battery (including a modified Irwin test, and qualitative assessment of the landing foot splay and fore/hind grip strength) and an automated test of motor activity. A detailed assessment for neurotoxicity effects was made on the basis of these measurements.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic or tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin and mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system. A score 0 was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink, and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was repeated at least 3 times for each animal. The distance between the two resulting ink spots for the hind and fore limbs was evaluated.
Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the forelimbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated as individual and mean data.
Quantitative assessment of motor activity was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, where DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters.
Data of all groups were evaluated for travelled distance (in 5-minute segments). The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls and comparing the treatment group.
No neurological examination was performed for Cohort 1B of F1 generation.
IN-LIFE PROCEDURES (F1 Animals)
Observation of offspring
Each litter of the parental females was examined as soon as possible after delivery (post-natal day 0 - PND 0: the day when the delivery finished) to establish the number and sex of pups, stillbirths, live births, and the presence of gross abnormalities (including cleft palate; subcutaneous haemorrhages; abnormal skin colour or texture; presence of umbilical cord; lack of milk in stomach; presence of dried secretions).
In addition, the first clinical examination of the neonates included a qualitative assessment of body temperature, state of activity and reaction to handling. Pups found dead on PND 0 or at a later time were examined for possible defects and cause of death.
Evidence of suckling was evaluated as of PND 0, based on the presence or lack of the milk in pup’s stomach.
On PND 0, the anogenital distance of each pup was measured.
All litters were checked daily for the number of viable and dead pups. Live pups were weighed with accuracy of 0.01 g at birth (PND 0) and on PND 4, 7, 14 and 21.
Routine clinical examinations were performed every day, detailed clinical observations were performed weekly. Observations could be performed more often if case-specific findings have been made at birth or during the lactation period. Signs noted included, but might not be limited to, external abnormalities, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity. Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also recorded.
On PND 4, litters were culled to yield, as nearly as possible, 5 males and 5 females per litter. Pups to be culled within each litter were selected at random. In litters of sufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less than 10. All culled (surplus) pups were subjected at least to necropsy with detailed macroscopic external and internal examination for any abnormalities.
The surplus pups at PND 4 were subjected to gross necropsy, and/or blood samples were taken for potential serum thyroid hormone (T4 and TSH) concentrations; one male and one female was sampled per litter (where litter size allowed sufficient remaining pups). Neonatal (PND 4) blood was pooled by litters for thyroid hormone analyses (to achieve the requested volume).
On PND 12 - 13, pups were examined for retention of nipples/areolae.
On PND 21, at least two males and two females from each litter were chosen at random for continuation of treatment. Selected pups were assigned to one of three sets designated for further treatment and evaluation. Littermates were group-housed by sex at least for the first week post-weaning wherever possible.
After weaning, the remaining F1 pups (not selected for cohorts) were submitted for a gross pathology examination as described in a later section. Blood was also collected for T4 and TSH analysis from weanlings (one male and one female sample per litter, wherever possible) subject to gross necropsy on PND 22 (i.e. pups not selected for cohorts).
During the weaning period, the pups only received test substance indirectly through the milk, until direct dosing commenced for them at weaning.
F1 animals’ continuation
The F1 pups selected for continuation were treated daily by oral administration from weaning (PND21). All animals were examined once daily for mortality and morbidity. Clinical observations (for checking behavioural changes and any overt signs of toxicity) was made daily and a detailed clinical observation (thorough physical examination) was conducted at least weekly on each animal. The body weight of each pup was recorded at least weekly, commencing from PND 21. Food consumption was evaluated weekly.
Females: Vaginal patency was evaluated daily for each F1 female (selected for cohorts) beginning PND 22 and any abnormality was noted. The body weight of each female was recorded on the day of vaginal patency. Oestrous cycle was monitored for two weeks following vaginal patency; care was taken to avoid induction of pseudopregnancy.
Males: All F1 males (selected for cohorts) were evaluated for balanopreputial separation daily beginning on PND 35 to detect if sexual maturation occurs early. The body weight of each male was recorded upon separation.
Selection of pups for post-weaning cohorts
At weaning (on PND 21) pups from all available litters of F1 generation (up to 24 pups/sex/group) were selected for further examinations and maintained until sexual maturation (unless earlier testing was required). Pups were selected randomly, with the exception that obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) were not normally included, as they were unlikely to be representative of the treatment group.
On PND 21, the selected F1 pups were randomly assigned to one cohorts of animals, as follows:
Cohort 1A = Reproductive/developmental toxicity testing
Cohort 1B = Reproductive/developmental toxicity testing
The F1 pups not selected for continuation were terminated after weaning on PND22 (except of 6 male and 6 female pups per dose group which were treated and kept for two weeks for replacement purpose in case of any pre-terminal death occurs). Pups were subjected to gross necropsy including an assessment of the reproductive organs. Blood was also collected for potential T4 and TSH analysis from weanlings (from 1 male and 1 female pups per litter). Sample collection was performed as described; no sample transfer and thyroid hormone analysis was conducted. Those samples will be discarded after the finalization of the study report.
For 10 pups per sex per group (from different litters) brain, spleen and thymus were weighed and retained in 10% neutral buffered formalin. In addition, mammary tissues for these male and female pups were retained in preservatives for optional microscopic analysis. No additional measurement was made but all those organs / tissues were archived for possible future analysis.
Cohort 1A: Reproductive/developmental toxicity testing
At least one male and one female/litter/group (up to 24 but at least 18/sex/group) were selected for primary assessment of effects upon the reproductive system and for assessment of general toxicity.
Animals were treated for 10 weeks after weaning (started on PND 21).
Vaginal smears were examined daily for all Cohort 1A females, after the onset of vaginal patency, until the first cornified smear was recorded, in order to determine the time interval between these two events. The stage of oestrous cycle was also checked on day of termination.
At termination (on Day 91, after 10 weeks treatment period), all procedures described in parental generation were followed (i.e. clinical pathology – including serum thyroid level assessment, sperm parameters in all males/oestrous cycle in all females, macroscopic examination, organ weight and tissue preservation, histopathology).
Cohort 1B: Reproductive/developmental toxicity testing
At least one male and one female/litter/group (up to 24 but at least 20 animals/sex/group) were selected with the priority for follow-up assessment of reproductive performance by mating F1 animals, when assessed. No such an assessment was deemed necessary based on the available results of the study at the time of decision making. These animals were also dedicated to obtaining additional histopathology data in cases of suspected reproductive or endocrine toxicants, or when results from Cohort 1A were equivocal. This was not necessary. No mating was performed using F1.
The following points would have triggered production of a second-generation:
- an adverse effect on fertility or fecundity of the parental generation,
- indications of abnormal sexual development of the F1 pups,
- adverse effects on F1 litter parameters and developmental landmarks,
- death or evidence of toxicity to the F1 pups pre-weaning, and
- equivocal effects on F1 parameters or unusual control data compared to historical background might have triggered a second generation.
The following organs were weighed, and corresponding tissues processed to the block stage: Vagina (not weighed), Uterus with cervix, Ovaries, Testes (at least one), Epididymides, Seminal vesicles and coagulating glands, Prostate, Pituitary, Vas deferens, Identified target organs.
As histopathology results from Cohort 1A were not equivocal, and no reproductive or endocrine toxicity was suspected, no additional histopathology of Cohort 1B animals was conducted.
Part 2 (Cohort 1A) animals were terminated on Day 91 of their treatment period. Prior to scheduled necropsy, clinical pathology examinations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all surviving animals. Samples were also taken for thyroid hormone measurements.
After an overnight period of food deprivation of animals (fasting), 3 blood samples were collected sublingual (for thyroid hormone analysis) or by cardiac puncture under pentobarbital anaesthesia, for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry. An additional sample was taken for thyroid hormone analysis (with K3-EDTA as anticoagulant, 1.6 mg/mL blood).
Urine samples were collected overnight (for approximately 16 hours) with no access to food, but with access to water, in individual metabolism cages.
All the procedures of Cohort 1A were followed for Cohort 1B (in order to provide additional samples if needed in case of equivocal results of Cohort 1A).

Postmortem examinations (parental animals):

Terminal procedures and macroscopic evaluation
Unscheduled deaths
Gross pathology was performed on every animal irrespective of the date of its death. Dead animals or moribund animals or animals euthanised for humane reasons were necropsied as early as possible after discovery, according to the relevant SOPs of the Test Facility. When necropsy was not an immediate action (in case of some found dead pups) the carcass was refrigerated (approximately 4 °C) until necropsy could be performed.
Dead pups were examined externally for gross abnormalities. Dead (intact) pups were necropsied with macroscopic examination in order to identify the probable cause of death.
Scheduled termination
Terminally (one day after the last treatment), sodium pentobarbital administered by intraperitoneal injection, followed by exsanguination was used for euthanasia. After exsanguination the external appearance was examined; cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
For all P0 females a vaginal smear was prepared and examined to determine the stage of the oestrous cycle at sacrifice as a possible aid for histopathology evaluation. The number of implantation sites and corpora lutea was recorded in all cohabited females as applicable.
At termination for all P0 males, after the organ-weight measurement of testis and epididymis, one of each organ was collected for sperm evaluation as described below. The other pair of the organs were preserved for histopathological examination.
Immediately after sacrifice, sperm from the cauda epididymis were collected for determination of motility and sperm morphology. For sperm motility, the percentage of progressively motile sperms were determined subjectively for 200 sperms for each male in duplicates, and the percentage of progressively motile sperms was determined subjectively. For measurement of sperm morphology, 200 sperm cells per sample (in duplicate slides, thus 400 cells in total) were evaluated from wet preparations and classified as either normal or abnormal (fusion, isolated heads, misshapen heads and/or tails).
Sperm motility for all animals was evaluated immediately after sacrifice. The smears for morphology were prepared and stored for later analysis, and also the testes and caput epididymides for homogenisation-resistant sperm counts. Analyses on these parameters were done on the Control and High dose groups only; based on the observed results examination of Low and Mid-dose groups were not deemed necessary.
Histopathology
For parental animals, in addition to the organs listed for organ-weight measurement, samples of peripheral nerve, muscle, spinal cord, eye plus optic nerve, gastrointestinal tract, urinary bladder, lung, trachea (with thyroid and parathyroid attached), bone marrow, vas deferens (males), mammary gland (males and females) and vagina were retained in suitable medium for histopathological examination.
The eyes with the optic nerve and the testes with epididymides were preserved in modified Davidson’s fixative, and all other organs in 10% buffered formalin solution.
The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
Detailed histological examination was performed as follows:
- retained organs in the Control (Group 1) and High-dose (Group 4) groups,
- all macroscopic findings (abnormalities), except of minor order from all animals
- retained reproductive organs (testes, epididymides, prostate, seminal vesicles with coagulation gland for males, and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Low-and Mid-dose groups suspected of reduced fertility (e.g., males failed to conceive / sire and females failed to deliver healthy offspring), or for which oestrus cyclicity or sperm number, motility, or morphology were affected.
Detailed testicular histopathological examination was conducted in order to identify treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cell, or sloughing of cells into the lumen. Examination of the epididymis included evaluation of the caput, corpus and cauda using longitudinal sectioning.
Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma. For females (for at least 10 females/dose group), a qualitative depletion of the primordial and small growing follicles as well as the corpora lutea in post-lactational ovary were evaluated.
The eyes with the optic nerve and the testes with epididymides were preserved in modified Davidson’s fixative; all other organs in 10% buffered formalin solution.

Postmortem examinations (offspring):

Necropsy and macroscopic examination were performed on all surviving animals of Cohort 1A at the end of treatment period (on Day 91), after the sample collection for clinical pathology was made. The animals were euthanised by exsanguination under pentobarbital anaesthesia similarly to the parental generation.
Prior to necropsy, the oestrus cycle of all females of Cohort 1A was determined by taking vaginal smears in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist the histological evaluation of oestrogen-sensitive tissues.
All the procedures for Cohort 1A were followed for animals of Cohort 1B.
Organ weight measurements
The following organs of surviving animals were trimmed of fat and weighed (parental generation and Cohort 1A of F1 generation) at termination:
With precision of at least 0.01g: Brain, Epididymides (total and cauda), Heart, Kidneys, Liver, Prostate (dorsolateral and ventral parts combined), Seminal vesicles with Coagulating glands, Spleen, Testes, Thymus, Uterus (including oviducts and cervix).
With precision of at least 0.001g: Adrenal glands, Ovaries, Thyroid with parathyroid glands (post-fixation), Pituitary.
Paired organs were weighed together. In case of necessity, for example if any significant difference in size was noted between paired organs, the individual weight of each organ was recorded. Absolute organ weights were measured; relative organ weights to the body and brain weights (if applicable) were calculated and reported.
On completion of the macroscopic examination, the following tissues and organs were retained from all Cohort 1A animals in suitable medium: Animal identification (Fixation and preservation only), Adrenals, Aorta, Brain, Epididymis, Eye with the optic nerve, Oesophagus, Femur with marrow, Heart, Kidney, Large intestine, Extraorbital lachrymal gland, Harderian gland, Liver, Lungs with bronchi, Lymph node, Ovary, Oviduct, Pancreas, Pituitary, Prostate, Salivary gland (including mandibular, sublingual and parotid glands), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, subcutis with mammary gland (inguinal), Skeletal muscle (quadriceps), Small intestine, Spinal cord, Spleen, Sternum with marrow, Stomach, Testis, Thymus, Thyroid with parathyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina
Vas deferens was also preserved for at least 20 males/group of Cohort 1A (only Control and High dose was evaluated at the first instance).
The retained tissues and organs were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed in Group 5 (Control) and Group 8 (High dose) and any animals found dead or euthanised pre-terminally during the study. In addition, any organs or tissues with macroscopic abnormalities (except minor changes) were subjected to histological examination from all groups.

Statistics:

Data capture and statistical methods - when activities were performed at the Test Sites - are reported in the appropriate phase reports. For Test Facility data and statistics, the following applies.
Data were recorded on the appropriate forms from the relevant SOPs of the Test Facility, then tabulated using the Microsoft Office Word and/or Excel, or collected using the software PROVANTIS v.9, as appropriate. Group means and standard deviations were calculated from numerical data obtained in the study.
The statistical evaluation of appropriate data (marked as † in the lists and tables) was performed with the statistical programme package of SAS 9.2 (when using Provantis) or by programme package SPSS PC+4.0 (SPSS Hungary Ltd., Budapest).
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree was applied automatically for statistical evaluation of continuous numerical data. The normality and heterogeneity of variance between groups was checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an ANOVA / ANCOVA (one-way analysis of (co)variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences, identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid, and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test, identifying differences of <0.05 or <0.01 as appropriate.

Reproductive indices:

see below in any other information on materials and methods

Offspring viability indices:

see below in any other information on materials and methods

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observation for found dead or pre-terminally euthanized animals are summarized in the mortality section. Noisy respiration was present mostly in the High dose group; this fact was related to the test item administration. Other clinical signs, not related to the test item treatment, were also detected for some terminally euthanized parental animals during the study as reported in the details section.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Three animals (one High dose male and female, and one Low dose female) were found dead during the study, three additional High dose females were pre-terminally euthanized. In case of the found dead animals, gavage accident was considered as a cause of death for the High dose and Low dose females, while a specific cause of death for High dose male was not determined. For the three pre-terminally euthanized females, inflammation of the cerebral/cerebellar meninges was considered as a major factor contributing to the clinical conditions and preterminal euthanasia. These findings were not attributed to the treatment with test item. More details can be found below.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No clear test item-related effect was noted on the mean body weight or body weight gain values of the parental animals (see also attached graphs and details section).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related effects in the mean daily food consumption in any test item-treated group when compared to the control. The summary is shown in a Table in the detail section.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

There were no test item-related effects in the mean water consumption in any test item-treated group when compared to the control. The summary is shown in the details section.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related effects were observed during visual inspection of eyes in any dose group. Besides, no detailed ophthalmoscopic examination was conducted for parental (P) adult animals.

Haematological findings:

no effects observed

Description (incidence and severity):

Ten male and 10 female parental animals from each group were selected for clinical pathology sampling and analysis at termination (on Day 121-124 in males and on PPD 22 in females).
Haematology
When compared to the controls, there were no differences in parental (P) males and females that could be considered toxicologically significant in the Low, Mid and High dose groups.
No statistically significant changes were recorded for any other parameters in the test item treated parental males and females when compared to control.
Coagulation parameters
There was no effect of the test item on blood clotting parameters.
No statistically significant or biologically relevant changes in the examined coagulation parameters (activated partial thromboplastin time, prothrombin time and fibrinogen) were recorded for any dose groups of parental (P) animals when compared to control animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in the serum chemistry parameters of the parental (P) generation. A summary of the selected parameters (where sporadic statistical differences were noted) is presented in the details section.

Endocrine findings:

no effects observed

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in the test item treated groups were observed in the urinalysis. A summary of the selected parameters is presented in the details section.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No test item effects on thyroid hormone levels were detected as summarised in the details section.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no effects on the F1 offspring viability, clinical signs, sexual development or at macroscopic observations in any dose groups. No significant differences between any of the dose groups and control were observed, neither in P(0) not in Cohort 1A animals.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

A test item related effect on fertility was seen in the High dose group for males, the mating process was not affected. No significant effect was observed in the Mid or Low dose male groups and also no dose dependency was observed. Thus, this finding may be seen as slight if not equivocal (see details section).
Sperm motility and morphology as well as number of sperms were examined in the study for parental males. There was an equivocal test item treatment-related effect in the High dose male of parental (F0) generation (summary is shown in details section), but in F1A and F1B males had completely normal sperm.

Reproductive performance:

no effects observed

Description (incidence and severity):

The mating indices were normal in all test item-treated groups (100%) for both males and females (see details in details section).
Test item administration was considered to have no impact on the duration of the mating period. The mean duration of mating was 3.54, 4.13, 4.04 and 3.55 days in Control, Low, Mid and High dose groups, respectively. All those values were in the normal range, and none of the differences compared to the control data were statistically significant.
No effect of test item treatment were noted during gestation period, parturition or the post-partum period (summary is presented in details section).
The mean length of gestation was 22.79, 22.95, 22.88 and 23.05 days in Control, Low, Mid and High dose groups, respectively. These values are well within the normal range for this strain of rat.

For details on results including tables please refer to attachment!

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

Remarks on result:

other: slight and equivocal, non-dose dependent effects, seen in male sperm morphology and slightly reduced male fertility in P0 high dose group was not observed in F1 generation and thus is considered not critical.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: based on systemic toxicity for parental generation

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

testes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Based on the external evaluation, all the pups in the study were normal, there were no effect of treatment.
In Cohort 1A and 1B animals noisy / laboured / gasping respiration was observed mostly for High dose animals. This fact was considered to be test item related (a common finding if a test item can cause local respiratory irritation after gavage, e.g. with acidic substances). The clinical signs recorded occasionally for some animals in the Mid and Low dose groups were not related to the test item treatment.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Overall, there were no treatment-related effects on the mortality or viability of pups on PND 0 until PND 21. All groups had survival index to PND 21 of >99% (see Tables in detail section).
In Cohort 1A two animals (one High dose male and one Mid dose female) were found dead during the study. Gavage accident was considered as a cause of death for the High dose male. A specific cause of death for Mid dose female was not determined. More details can be found in the pathology section. Also, for Cohort 1B a total of ten test item treated animals were found dead during the study, but no clearly test item-related mortality was observed in this cohort during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean individual birth weights (on PND 0) of male and female live-born pups delivered by treated dams as well as their body weight at weaning (PND 21) were all within the normal range with no significant difference from the control group (see Table in details section), similar situation was observed for body weight gain.
No test item effect was noted on body weight or body weight gain in animals of Cohorts 1A or 1B (see also Figures attached).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In case of Cohort 1A animals (males and females), no statistically significant difference in the daily mean food consumption of any test item group was detected compared to control when calculated for the complete treatment period (PND21-90). The observed minor, statistically not significant changes (decreases or increases) in some periods were considered as biological variability. A summary table is shown in the details section. No test item effect was noted on food consumption in Cohort 1B.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related effects were observed during visual inspection of eyes in any dose group. Besides, no detailed ophthalmoscopic examination was conducted for cohort 1A or 1B animals.

Haematological findings:

no effects observed

Description (incidence and severity):

When compared to the controls, there were no differences in Cohort 1A and 1B males and females that could be considered toxicologically significant in the Low, Mid and High dose groups as documented in the details section.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

All male and female Cohort 1A animals from each group were used for clinical pathology sampling and analysis at termination (on PND 121).
Haematology
When compared to the controls, there were no differences in Cohort 1A or 1B males and females that could be considered toxicologically significant in the Low, Mid and High dose groups as documented in the details section.

Coagulation parameters
There was no effect of the test item on blood clotting parameters.
No statistically significant or biologically relevant changes in activated partial thromboplastin time and prothrombin time were recorded for any dose groups of Cohort 1A or 1B animals.

Clinical chemistry
There were no test item-related changes on the serum chemistry parameters of Cohort 1A or 1B animals. Summary of the selected parameters is seen in the details section.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in the test item exposed groups were observed in the urinalysis of Cohort 1A or 1B (see also details section).

Sexual maturation:

no effects observed

Description (incidence and severity):

Additional parameters (balanopreputial separation for males, vaginal opening and oestrus cycle for females) were also examined to follow the sexual development relative to the body weight development of F1 (Cohort 1A and 1B) weanlings. None of the parameters investigated showed significant differences between test item treated animals in dose groups and control groups.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance was measured on PND 0 in new-born F1 pups. There was no test item effect on this parameter in the test item-treated males and females of F1 generation (The summary table is shown in the details section).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No difference regarding nipple retention between control and the Low, Medium and High dose groups were observed.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Terminal body weights of males and females were not significantly different between the groups of Cohort 1A. There were no significant differences considered to be treatment-related in the test item treated animals (males and females) when compared to control data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Found dead animals
One High dose male (#8005) and one Mid dose female (#7516) were found dead on PND 46 and 44, respectively. Gavage accident was considered as a cause of death for the High dose male. A specific cause of death for Mid dose female was not determined.
Terminally euthanized animals
Macroscopic evaluation
There were no test item related macroscopic findings observed at Low, Mid and High dose levels (50, 150 and 500 mg/kg bw/day, respectively) in Cohort 1A.
Microscopic evaluation
There were no test item-related microscopic findings observed at a High dose level of 500 mg/kg bw/day in Cohort 1A.
All changes in test item given animals were seen without meaningful differences in severity and incidence and considered to be incidental or a common background.

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Sperm analysis
Sperm motility and morphology as well as number of sperms were examined in the study for Cohort 1A and 1B males. No treatment-related effect was seen in any dose groups of this cohort (summary is shown in details section).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no test item-related effects in the neurological assessment.
There were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item-treated groups in the Irwin test. Investigations on grip strength, locomotor activity measurements (SMART) and auditory startle reflex assessment also showed no significant difference to control (see also table in details section).

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in the test item treated groups compared to control were observed in the thyroid hormone analysis results or thyroid weight of Cohort 1A animals as tabulated in the details section.

For details on results including tables please refer to attachment!

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1B)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Lowest effective dose / conc.:

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

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

A daily administration of Sulfamic acid by oral gavage to Crl:WI (Wistar) rats at dose levels of 50, 150 and 500 mg/kg bw/day for a minimum of 17 weeks (Parental generation), followed by F1 generation pups selected for daily treatment by oral administration from weaning and terminated after the end of their treatment period (Cohort 1A), did not produce any macroscopic and microscopic test item-related findings.
There were no changes seen in the study that would suggest a generational effect of the test item on any of the evaluated parameters.

Executive summary:

The purpose of this study was to conduct an extended one-generation reproduction study as proposed by ECHA for this specific project, based on the OECD Guidelines for Testing of Chemicals No. 443 (2018) and as requested by the European Chemical Agency (ECHA) decision letter. The main objective of the Extended One-Generation Reproductive Toxicity Study was to evaluate specific life stages not covered by other types of toxicity studies and test for effects that may occur as a result of pre- and postnatal chemical exposure.

Male and female Wistar rats were exposed by the oral route for 10 weeks pre-mating and then during the mating/post-mating periods. Females were further exposed throughout gestation and up to and including postpartum/lactation day (PPD) 22. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or weekly detailed observation of clinical signs, at least weekly body weight and food consumption and clinical pathology evaluation (including haematology, coagulation, clinical chemistry, urinalysis and thyroid hormone measurement). Water consumption was also measured in all phases of the study. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals. Litter size was standardised on post-natal day (PND) 4; viability, clinical signs and development (physical and sexual) were evaluated in the F1 offspring until weaning on PND 21.

F1 pups were selected for one cohort at weaning, non-selected pups were terminated on PND22. The test item exposure of the selected pups continued by the oral route after weaning until termination. Animals of Cohort 1 (1A and 1B) were dedicated to reproductive/developmental toxicity testing. For Cohort 1 animals the same parameters were monitored as for parental animals. Clinical pathology examinations (including haematology, coagulation, clinical chemistry, urinalysis and thyroid hormone measurement) were conducted for Cohort 1A prior to necropsy on PND 91 (plus recovery animals from Cohort 1B). Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed in the period of Days 64-69 of the exposure for Cohort 1A. Startle response was also evaluated for Cohort 1A animals.

At termination, necropsy with macroscopic examination was performed for parental and selected F1 animals. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives. For the parental and selected F1 animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

 

Results

Overall, based on the performed analytical verification measurements (by titration method and supporting Raman spectroscopy data), the test item formulations were considered to be suitable for the study purposes.

In summary, daily oral administration of Sulfamic acid to Wistar rats at dose levels of 50 mg/kg bw/day (Low-dose group), 150 mg/kg bw/day (Mid-dose group) and 500 mg/kg bw/day (High-dose group) showed the following results under the conditions of this Extended One Generation Reproductive Toxicity Study.

 

Parental (F0) generation

No clearly test item-related mortality was observed in the parental animals. Three animals (one High dose male and female, and one Low dose female) were found dead, three additional High dose females were pre-terminally euthanized. Gavage accident was considered as a cause of death for the found dead females, while a specific cause of death for High dose male was not determined. For the three pre-terminally euthanized females, inflammation of the cerebral/cerebellar meninges (unrelated to treatment) was considered as a major factor contributing to the clinical conditions and preterminal euthanasia. These findings were not attributed to the treatment with test item.

Noisy respiration was present mostly in the High dose group, this was a local irritation response, related to the test item administration.

No clear test item-related effect was noted on the mean body weight or body weight gain values, although the body weight gain values of High dose females were more than 10% lower than control in the pre-mating period or gestation period, but no statistical significance was gained.

There were no test item-related effects in the mean daily food or water consumption of parental animals.

Test item related effect was seen on fertility in the High dose group. No test item-related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period in Mid and Low dose groups when compared to control.

There was an equivocal effect on sperm morphology in the High dose group.

No test item effect on thyroid hormone levels were detected.

There were no effects on the F1 offspring viability, clinical signs, sexual development or at macroscopic observations in any dose groups.

There were no test item related effects on clinical pathology parameters (haematology, coagulation, clinical chemical or urinalysis.

Increased liver weights in the High dose group (in the absence of histopathological correlate) suggesting an adaptive mild hypertrophy, thus a non-adverse test item related effect.

There were no test item-related macroscopic or microscopic findings observed at Low, Mid and High dose groups of the parental generation.

 

Cohorts 1A

No clearly test item-related mortality was observed during the study. Two animals (one High dose male and one Mid dose female) were found dead during the study. Gavage accident was considered as a cause of death for the High dose male. A specific cause of death for Mid dose female was not determined.

No test item effect was noted on body weight, body weight gain or food consumption in this cohort.

There were no test item-related effects in the neurological assessment.

There were no effects on clinical pathology parameters (haematology, coagulation, clinical chemical or urinalysis).

There were no effects in sperm analysis of the male animals of this cohort.

There were no test item-related adverse differences in organ weights, and no test item related macroscopic or microscopic findings were observed in any dose groups.

A quantitative assessment of primordial follicles and corpora lutea conducted in females did not show any meaningful differences in total number of primordial follicles and corpora lutea between Control and High dose females.

 

Cohort 1B

No clearly test item-related mortality was observed in this cohort. A total of ten treated animals were found dead: one Low dose male, two Mid dose females, three High dose males and four High dose females. No test item-related macroscopic findings were observed in these animals, for one High dose female a gavage accident was confirmed.

Noisy / gasping / laboured respiration was observed mostly for High dose animals. This was considered to be a local irritation response, test item related.

No test item effect was noted on body weight, body weight gain or food consumption in this cohort.

No test item effect was noted on sexual development (balanopreputial separation for males, vaginal opening and oestrus cycle for females).

There were no effects on clinical pathology parameters (haematology, coagulation, clinical chemical or urinalysis).

There were no test item-related necropsy findings observed in the animals of Cohort 1B, and no test item effect was identified during sperm analysis.

There were no test item-related organ weight changes and macroscopic findings observed at the Low, Mid and High dose levels of Cohort 1B.

No treatment-related effect was seen in sperm analysis of the male animals of this cohort.

 

All generations

Clinical signs of local respiratory irritation were observed in all cohorts at the High dose level. There were no clear effects on body weight, weight gain or food intake. Clinical pathology showed no clear effects of test item. Organ weights indicated a non-adverse mild hypertrophy at the High dose, particularly in females. No clear effect was concluded on the mating and reproduction of females at any dose level. High dose males in the F0 generation appeared to have a reduced fertility (no dose dependency was observed), and an equivocal observation of a higher rate of morphologically abnormal sperm were seen, but in F1A and F1B males had completely normal sperm. Hence, this observation made in the parental high dose group was not considered critical.

A daily administration of Sulfamic acid by oral gavage to Crl:WI (Wistar) rats at dose levels of 50, 150 and 500 mg/kg bw/day for a minimum of 17 weeks (Parental generation), followed by F1 generation pups selected for daily treatment by oral administration from weaning and terminated after the end of their treatment period (Cohort 1A), did not produce any macroscopic and microscopic test item-related findings.

There were no changes seen in the study that would suggest a generational effect of the test item on any of the evaluated parameters.

In conclusion, under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) values for Sulfamic acid was considered to be as follows:

Based on test item effect on body weight, the NOAEL for systemic toxicity of parental generation: 500 mg/kg bw/day.

Based on the effect on the reproductive parameters of parental animals (apparent lower male fertility at the High dose, and equivocal morphological sperm observations (restricted to the F0 generation), the NOAEL for reproductive toxicity of parental generation: 150 mg/kg bw/day.

Due to lack of any test item effect on the body weight or food consumption of F1 generation, the NOAEL for physical development of F1 pups: 500 mg/kg bw/day.

Due to lack of any test item effect on the sexual development of F1 generation, the NOAEL for sexual development of F1 pups: 500 mg/kg bw/day.

 

No indication of any neurotoxic effect of the test item was indicated in the study.

No indication of any immunotoxic effect of the test item was indicated in the study.

No endocrine disruptor effect of the test item was indicated in the study.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No developmental toxicity was observed in an OECD 414 study in the absence of maternal toxicity. Likewise, also in an OECD 443 study with rats, no developmental effects were observed up to 500 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant OECD414 study without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Rat / CD / Crl:CD (SD)

Details on test animals or test system and environmental conditions:

Mature female CD® rats were used for this experiment, a strain bred by Charles River Laboratories, Research Models and Services, Germany GmbH.
Each animal was given a thorough examination. Animals were judged to be healthy on the basis of general observations (physical examination). Health checks were performed on the day of delivery and at the beginning of matings. The spare animals were retained to replace any rat showing signs of illness. No replacements had to be carried out.
Body weight (on day 0 of gestation): 196.2 - 240.8 g
Age (on day 0 of gestation): 60 days
For identification, each rat received a continuous number between 1 and 100: a pattern of points was set on paws and/or tail by tattoo. Additionally, the animal cages were labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose level, date of conception and dates of administration.
Diet: Commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Ger-many, see Appendix 2: Composition of the Diet) served as food. This food was offered daily ad libitum. Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year. Certificates of analysis of the composition and for contaminants were provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. The room temperature was 22 °C ±3 °C (maximum range) and the relative humidity 55% ±15% (maximum range). No values outside the maximum range were noted during the course of the study. Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were cleaned and changed once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a ear by LUFA-ITL.
The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle.
Drinking water: Drinking water was offered ad libitum. Samples of drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on drinking water 2001].
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on Drinking Water 2001].

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Route of administration: Oral by gavage
Frequency of administration: Once daily
Treatment period: Day 6 to 19 of gestation
Vehicle: Water for injection
Administration volume: 10 mL/kg b.w./day
Selection of route of administration: According to OECD guideline 414
The test item formulations were freshly prepared every day. The test item was dissolved in the vehicle to the appropriate concentration and was administered orally at a constant volume once daily. The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume dailly in the same way.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item formulations, samples of approximately 5 mL were taken at the following times and stored at -20 °C or colder until analysis:
At study initiation: Analysis of stability and concentration was done immediately after preparation of the formulation as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples /dose level group; groups 2 - 4).
At study termination (at a time when the majority of animals was dosed) during treatment with the test item always before administration to the last animal of the group (1 sample/group).
The samples were labelled with the study number, species, type of sample, concentration, test day, sampling time and date.
The concentration of Sulphamic Acid in the test item formulation samples was determined by measuring the amount of a NaOH solution (0.01M, 0.05M or 0.15M) which was necessary to cause the colour change of the pH indicator (Phenolpthalein) in one mL test item formulation.
The following calculation was used: One mL 0.01 M NaOH is necessary to cause the colour change of the pH indicator in a solution of 1 mL test item formulation containing 0.971 mg Sulphamic Acid. Five titrations were performed from each sample of test item formulation.
The measured actual concentrations of the test item in the test item vehicle mixtures were between 102.0% and 105.7% of the nominal concentrations, indicating correctly prepared formulations which were stable at room temperature for at least 24h.

Details on mating procedure:

Four groups of pregnant rats were formed from matings, which were carried out on a daily basis. Vaginal lavages were taken each morning. When positive, the animals were assigned to the test groups by mating day using a Provantis (Version 8.2.0.8) generated randomization.
Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant (3 control animals out of 25 control animals) were excluded from the analysis of the results and replaced by spare animals. The number of non-pregnant animals of this study was within the normal range of variation. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.

Duration of treatment / exposure:

Pregnant rats were treated with the test item starting from the 6th and lasting until the 19th day of pregnancy (the 'critical' phase of organogenesis and fetal development).

Frequency of treatment:

Once daily

Duration of test:

Start of study
First mating results: February 25, 2014
First administration: March 03, 2014
Study termination
End of the in-life part: March 20, 2014

Remarks:

Doses / Concentrations:
60 mg/kg b.w./day
Basis:
nominal in water

Remarks:

Doses / Concentrations:
200 mg/kg b.w./day
Basis:
nominal in water

Remarks:

Doses / Concentrations:
600 mg/kg b.w./day
Basis:
nominal in water

No. of animals per sex per dose:

25 animals per dose, 20 of them to be evaluated

Control animals:

yes, concurrent vehicle

Details on study design:

Four groups of pregnant rats were formed from matings, which were carried out on a daily basis. Vaginal lavages were taken each morning. When positive, the animals were assigned to the test groups by mating day using a Provantis (Version 8.2.0.8) generated randomization.
Justification for dose selection: The dose levels were selected in agreement with the Sponsor based on available toxicity data and a dose-range finding Study (no. 30799) performed at LPT. In the dose-range finding study, Sulphamic Acid was administered to female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day orally, by gavage, once daily for 14 days. Oral treatment with 100 mg Sulphamic Acid/kg b.w./day for 14 days did not cause any signs of systemic intolerance reactions. At 300 mg Sulphamic Acid/kg b.w./day, breathing sounds were recorded for one of three intermediate dose females on two test days. Treatment with 1000 mg Sulphamic acid/kg b.w./day caused slightly or moderately reduced motility, breathing sounds or laboured breathing, increased salivation and/or a haemorrhagic nose/snout. In addition, transient reductions were noted for body weight, body weight gain and food intake. One high dose female was found dead on test day 3. Necropsy after premature death revealed gastro-intestinal lesions (multiple haemorrhagic foci at the fundus region of the stomach, inflated intestines) and hepatic changes (liver partly pale). No test item-related findings were noted for the consistency of faeces in the three females each treated with 100 or 300 mg Sulphamic acid/kg b.w./day or in the two surviving females treated with 1000 mg Sulphamic acid/kg b.w./day. The visual appraisal of the drinking water consumption revealed no test item-related influence. Macroscopic inspection during necropsy revealed no test item-related gross pathology changes. Normal values compared to the control were noted for relative and absolute organ weights of liver, kidneys and ovaries.

Maternal examinations:

Clinical signs: Individual animals were observed daily for behaviour, external appearance and nature of the faeces. Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Viability: Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday. Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.
Body weight: The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20). Furthermore the net weight change from day 6 is given. These values are stated in the report. These measurements were also used for calculating the daily amount of test item to be administered.
Food and drinking water consumption: The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day. The relative food consumption (g/kg b.w./day) was calculated using the following formula: Daily food consumption [g/kg b.w./day] = Total food intake in g x 1000/Body weight in g
Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.

Ovaries and uterine content:

Necropsy: On the 20th day of gestation, the rats were laparotomised under ether narcosis. The ovaries and the uteri of the dams were removed; the uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae was determined
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) Fetuses were inspected externally for damages, especially for malformations .
(i) The fetuses were sacrificed by an ether atmosphere.
(j) Examination of fetuses and determination of number and kind of retardations, variations or malformations: 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations). The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON. The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.

Fetal examinations:

Evaluation / parameters
Corpora lutea
- number per dam
- absolute number per group
- mean per group
Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
Resorptions
- number per dam, % per litter
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- early resorptions <2 mm, number and % per litter
- Late resorptions >2 mm, number and % per litter
- % litters with resorptions per group.
Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- litter mean per group
- litter mean per sex and group
Weight of fetuses
- individual data per fetus
- mean per litter
- mean per group
- litter mean per group
- litter mean per sex and group
Fetuses
- number and % per dam (alive)
- number and % per dam (dead)
- number of fetuses per sex and dam
- sex ratio per litter
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- mean % per sex and group
Runts
- number per dam
- mean per group
Malformed fetuses
- individual data per fetus
- type of malformation: fetal and litter incidence
- number of affected fetuses per group [%] (fetuses affected by several changes counted as one fetal incidence)
Fetuses with variations
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- Number of affected fetuses per group [%] (fetuses affected by several changes counted as one fetal incidence)
Fetuses with retardations
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- number of affected fetuses per group [%] (fetuses affected by several changes counted as one fetal incidence)
Pre-implantation loss (mean % per group; values per group)
Post-implantation loss (mean % per group; values per group)

Statistics:

The following data were captured or calculated by the the departmental computerized system (Provantis integrated preclinical software, version 8.2.0.8, Instem LSS Ltd): Clinical signs, body weight, body weight gain, food consumption, gravid uterus weight, carcass weight and the net and absolute weight change from day 6 until laparotomy. Raw data not fully compatible with the computerized sytem (e.g. reproduction data, skeletal and soft tissue examination) were maintained on paper according to the appropriate SOPs.
Statistical analyses of the parametrical values, captured or calculated by Provantis, were done by Provantis using the following settings: Analysis of normal distribution and homogeneity of variances was perfromed by using the SHAPIRO-WILKS test and the BARTLETT test. Data not normally distributed or with heterogenous variances between the groups were stepwise log- or rank-transformed. One-way analysis of variance (ANOVA) was performed with non-transformed or log-transformed data. The KRUSKAL-WALLIS test was used for rank-transformed data. In case of significant differences (found by ANOVA or KRUSKAL-WALLIS test), inter-group comparisons with the control group were made by parametric or non-parametric DUNNETT multiple comparison tests (p ≤ 0.05 and p ≤ 0.01). Parametrical values not captured by Provantis (e.g. number and weight of the fetuses) were analysed by the DUNNETT test (p ≤ 0.05 and p ≤ 0.01). Prior to the DUNNETT test homogeneity of variances was tested using the BARTLETT test. In case of heterogeneity of variances, the STUDENT's t-test was carried out (p ≤ 0.05 and p ≤ 0.01). Statistical analyses of non-parametrical data (e.g. resorption-, malformation-, variation and retardation rates) were performed using the following settings: FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01). Significantly different data are indicated in the summary tables of the result sections of the report.

Historical control data:

LPT Background Data range of mean values per group (n=56 control or n= 143 test item groups; Data taken from 2000 - 2014) were considered in the evaluation of test data and results.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At 600 mg Sulphamic Acid/kg b.w./day several dams revealed signs of toxicity in form of behavioural changes and a reduction in the mean body weight and in food consumption in comparison to the control group.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
An increased number of resorptions, a slightly reduced fetal body weight and delayed ossifications for the os ischii, the sacral and caudal vertebral bodies and the metatarsalia were noted at the materno-toxic dose level of 600 mg Sulphamic Acid/kg b.w./day.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Examination of the dams:

Mortality: None of the dams died prematurely.

Clinical signs: At 600 mg test item/kg b.w./day 11 of 20 dams revealed several signs of clinical toxicity like piloerection, an extreme yellow discoloured urine, salivation and an decreased or increased water consumption.

Group 4: Dams with signs of toxicity(+: sign observed (.): number of test days)
Dam no.	Piloerection	Extreme yellow discoloured urine	Salivation	Water consumption decreased	Water consumption increased
77			+ (6)	+ (6)
78			+ (7)		+ (3)
79					+ (3)
80					+ (8)
81		+ (7)	+ (7)	+ (9)	+ (1)
82			+ (4)
85		+ (10)			+ (10)
86			+ (3)
91	+ (7)	+ (8)		+ (10)
92	+ (2)	+ (4)	+ (9)	+ (9)
93	+ (2)		+ (6)

 

body weight gain: Treatment with 600 mg test item/kg b.w./day led to a transient reduction in body weight gain for the dams of the high dose group between gestation day 6 and 9, leading to a reduced body weight by 4.4% (statistically not significant) in comparison to the control group on the day of laparotomy. The net weight change (body weight gain without the uterus) from the start of treatment until laparotomy was decreased by 55.7% for the dams of the high dose group (600 mg test item/kg b.w./day) in comparison to the control group (statistically not significant).

Food consumption: 600 mg test item/kg b.w./day caused a reduced food intake after the start of treatment between gestation days 6 and 12 by max. 19.1% (statistically not significant).

Drinking water consumption: A decreased water consumption was noted during the daily visual appraisal for 4 of 20 dams and an increased water consumption for 5 of 20 dams of the high dose group (600 mg test item/kg b.w./day).

Necropsy findings: No changes were noted during internal inspection at laparotomy.

Uterus and carcass weights: At 600 mg test item/kg b.w./day a slight reduction in the gravid uterus weight by 11.2% (statistically not significant) was noted.

Examination of the fetuses:

An increased number of resorptions was noted in the high dose group (600 mg test item/kg b.w./day), leading to an increased post-implantation loss and a statistically significantly (p ≤ 0.05) increased ratio of resorptions to implantation sites.

Parameter		Mean number of resorption per dam		LPT Background Data range of mean values per group (n=56 control or n= 143 test item groups; Data taken from 2000 - 2014)
Resorptions per dam		Group 1:	0.4	0.0 - 1.8 0.0 - 6.4	(Control) (test item groups)
		Group 2:	0.3
		Group 3:	0.4
		Group 4:	0.9*
*/**:	(p ≤ 0.05 / p ≤ 0.01) Chi^2t-test

 

Parameter					Group 1 Control (n=20)	Group 2 60 mg/kg (n=20)	Group 3 200 mg/kg (n=20)	Group 4 600 mg/kg (n=20)
Corpora lutea			total per dam		284 14.2	275 13.8	281 14.1	277 13.9
Implantation sites •1			total per dam		280 14.0	268 13.4	272 13.6	270 #1 13.5
Resorptions •2			total per dam		8 0.4	5 0.3	7 0.4	17* #2 0.9
Early resorptions •2			total per dam		6 0.3	4 0.2	6 0.3	11 0.6
Late resorptions •2			total per dam		2 0.1	1 0.1	1 0.1	6 #1 0.3
Live fetuses •3			total per dam		272 13.6	263 13.2	265 13.3	254 12.7
Dead fetuses at laparotomy			total		0	0	0	0
Pre-implantation loss			mean %		1.4	2.6	3.1	2.0
Post-implantation loss			mean %		2.8	1.9	2.8	6.4
	*	Significantly different from the controls at p ≤ 0.05, Chi^2test.
	**	Significantly different from the controls at p ≤ 0.01, Chi^2test.
	#1	including twins
	#2	mostly due to dam no. 88 with 6 resorptions (1 early + 5 late resorptions) and the number of resorptions per dam is still in the range of background data (0.0 - 1.8).
	For the statistical analyses of the above mentioned parameters of reproduction the following comparisons were performed:
	•1	comparing the values of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group using the Chi^2test. This comparison is described as pre-implantation loss .
	•2	comparing the values of resorptions/implantation sites of the test group with the ratio of resorptions/implantation sites of the control group using the Chi^2test.
	•3	comparing the values of live fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group using the Chi^2test. This comparison is described as post-implantation loss.

 

Mortality: No dead fetuses were noted in the study.

Sex distribution: No test item-related differences were noted.

Fetal weights: 600 mg test item/kg b.w./day resulted in a slight reduction in fetal body weight by 5.9%.

 

Parameter	Weight of the fetuses (g)		LPT Background Data#    range of mean values per group (n=56 control or n= 143 test item groups; Data taken from 2000 - 2014)
Male and female fetuses together	Group 1:	3.4	3.2 - 4.0	(Control)
	Group 2:	3.5	3.1 - 4.0	(test item groups)
	Group 3:	3.6 **	3.1 - 4.0	(test item groups)
	Group 4:	3.2	3.1 - 4.0	(test item groups)
Male fetuses	Group 1:	3.5	3.2 - 4.1	(Control)
	Group 2:	3.6	3.1 - 4.2	(test item groups)
	Group 3:	3.6	3.1 - 4.2	(test item groups)
	Group 4:	3.3	3.1 - 4.2	(test item groups)
Female fetuses	Group 1:	3.3	3.1 - 3.8	(Control)
	Group 2:	3.4	3.0 - 3.9	(test item groups)
	Group 3:	3.5 **	3.0 - 3.9	(test item groups)
	Group 4:	3.1	3.0 - 3.9	(test item groups)
#: not audited by QAU
*/**: (p ≤ 0.05 / p ≤ 0.01) Dunnett or Student's t-test

Placental weights: No test item-related differences were noted.

Runts: 2 runts were noted in the control group and no runts in the treatment groups.

Malformations: No test item-related malformations were noted in the fetuses during external / internal examination, skeletal examination (according to DAWSON) and during soft tissue evaluation (according to WILSON).

Variations: No test item-related variations were noted in the fetuses during external / internal examination, skeletal examination (according to DAWSON) and during soft tissue evaluation (according to WILSON).

Skeletal examination of the foetuses: No malformations were noted during skeletal examinations of the fetuses according to DAWSON in the control group and in any of the treatment groups.

Unclassified external observations: The observation of twins (5-1 and 5-2) from dam no. 92 of the high dose group (600 mg Sulphamic Acid/kg b.w./day) was considered as an unclassified external observation.

Retardations: At 600 mg test item/kg b.w./day the examination of the skeleton according to DAWSON revealed test item-related increased fetal incidences (statistically significant at p ≤ 0.05 or p ≤ 0.01) for the following parameters: absence of ossification in metatarsalia 2-5, caudal vertebral bodies (all bodies unossified), os ischii incompletely ossified or unossified, sacral vertebral body/bodies unossified.

Parameter		Group values observed in this study (fetal incidences in %)		LPT Background Data# range of mean values per group (fetal incidence in mean %) (n=55 control or n= 141 test item groups; Data taken from 2000 - 2014)
Absence of ossification in metatarsalia		Group 1:	8.8	0.0 - 42.3 0.0 - 37.6	(Control) (test item groups)
		Group 2:	4.5
		Group 3:	9.0
		Group 4:	18.9*
Caudal vertebral bodies, all bodies unossified		Group 1:	16.2	0.0 - 37.4 0.0 - 29.6	(Control) (test item groups)
		Group 2:	22.7
		Group 3:	16.5
		Group 4:	40.9**
Os ischii incompletely ossified		Group 1:	2.2	0.0 - 2.5 0.0 - 5.3	(Control) (test item groups)
		Group 2:	5.3
		Group 3:	1.5
		Group 4:	7.1*
Os ischii unossified		Group 1:	7.4	0.0 - 14.0 0.0 - 7.6	(Control) (test item groups)
		Group 2:	7.6
		Group 3:	6.0
		Group 4:	18.1**
Sacral vertebral body/bodies unossified		Group 1:	6.6	0.0 - 20.8 0.0 - 12.4	(Control) (test item groups)
		Group 2:	8.3
		Group 3:	7.5
		Group 4:	23.6**
Hyoid unossified		Group 1:	71.3	0.0 - 89.4 0.0 - 93.7	(Control) (test item groups)
		Group 2:	64.4
		Group 3:	56.4**
		Group 4:	59.8*
Skull incomplete ossification		Group 1:	71.3	1.4 - 71.3 1.4 - 79.5	(Control) (test item groups)
		Group 2:	79.5
		Group 3:	59.4*
		Group 4:	74.8
Sternebra(e) incompletely ossified		Group 1:	7.4	5.5 - 86.2 0.0 - 91.4	(Control) (test item groups)
		Group 2:	17.4**
		Group 3:	16.5*
		Group 4:	11.0
Sternebra(e) unossified		Group 1:	91.9	5.8 - 91.9 1.2 - 88.5	(Control) (test item groups)
		Group 2:	84.8
		Group 3:	80.5**
		Group 4:	86.6
Thoracic vertebral body/bodies dumbbell-shaped		Group 1:	27.9	0.0 - 27.9 0.0 - 43.2	(Control) (test item groups)
		Group 2:	43.2**
		Group 3:	36.8
		Group 4:	29.9
#:	not audited by QAU
*/**:	(p ≤ 0.05 / p ≤ 0.01) Fisher or Chi^2- test

The statistically significant changes listed in the table above are not considered to be test item-related due to the following reasons:

Hyoid unossified, skull incomplete ossification, sternebra(e) unossified: A decreased incidence of unossified parts of the skeletal in comparison to the control group is not an adverse effect.

Sternebra(e) incompletely ossified, thoracic vertebral body/bodies dumbbell-shaped: The increase in the fetal incidence is restricted to the low and/or the intermediate dose group, whereas no statistically significant differences between the high dose group and the control group were noted.

No malformations or test item-related variations were noted during the external macroscopic examination, the macroscopic internal examination for gross changes of internal organs, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON for the fetuses of the dams treated with 60, 200 or 600 mg Sulphamic Acid/kg b.w./day, p.o.

At the materno-toxic dose level of 600 mg Sulphamic Acid/kg b.w./day, p.o. the skeletal examination according to DAWSON revealed retardations in the form of delayed ossifications of the os ischii the sacral and caudal vertebral bodies and the metatarsalia. Delayed ossification has been reported as related to maternal toxicity, especially in case of the phalanges, sternebrae nos. 5/6, the cervical, thoracic, sacral and caudal vertebral centra and the calvarium (Carney and Kimmel, 2007).

 

Number of  fetuses with  malformations	Group 1 Control	Group 2 60 mg Sulphamic Acid/kg	Group 3 200 mg Sulphamic Acid/kg	Group 4 600 mg Sulphamic Acid/kg
external malformations  fetal incidence N                        %	1 0.4	0 0.0	0 0.0	1 0.4
external malformations  litter incidence N                        %	1 5.0	0 0.0	0 0.0	1 5.0
internal malformations  fetal incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
internal malformations  litter incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
skeletal malformations  fetal incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
skeletal malformations  litter incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
soft tissue malformations  fetal incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
soft tissue malformations  litter incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
external variations  fetal incidence N                        %	0 0.3	0 0.0	0 0.0	0 0.0
external variations  litter incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
internal variations  fetal incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
internal variations  litter incidence N                        %	0 0.0	0 0.0	0 0.0	0 0.0
skeletal variations  fetal incidence N                        %	0 0.0	3 2.3	3 2.3	1 0.8
skeletal variations  litter incidence N                        %	6 30.0	2 10.0	2 10.0	1 5.0
soft tissue variations  fetal incidence N                        %	0 0.0	3 2.3	3 2.3	1 0.8
soft tissue variations  litter incidence N                        %	0 00.0	2 10.0	2 10.0	1 5.0
skeletal retardations  fetal incidence N                        %	136 100.0	132 100.0	133 100.0	127 100.0
skeletal retardations  litter incidence N                        %	20 100.0	20 100.0	20 100.0	20 100.0

 

Conclusions:

Based on the findings of this OECD414 developmental toxicity study with sulphamic acid, no developmental toxicity in the absence of maternal toxicity was observed. Thus, sulphamic acid is not considered causing developmental toxicity in rats.

Executive summary:

In this rat embryotoxicity study, the test item sulphamic acid was administered orally to female rats at dose levels of 60, 200 or 600 mg/kg b.w./day from the 6th to 19th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 200 mg sulphamic acid/kg b.w./day for the dams. At 600 mg sulphamic acid/kg b.w./day several dams revealed signs of toxicity in form of behavioural changes and a reduction in the mean body weight and in food consumption in comparison to the control group. Necropsy revealed no changes for the dams of all treatment groups.  

The no-observed-adverse effect level (NOAEL) for the fetal organism was also at 200 mg sulphamic acid/kg b.w./day.

An increased number of resorptions, a slightly reduced fetal body weight and delayed ossifications for the os ischii, the sacral and caudal vertebral bodies and the metatarsalia were noted at the materno-toxic dose level of 600 mg sulphamic acid/kg b.w./day. No dead fetuses, no malformations and no test item-related variations were noted at any of the tested dose levels.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In an OECD 414 study with rats investigating embryotoxicity, the test item sulphamic acid was administered orally to female rats at dose levels of 60, 200 or 600 mg/kg bw/day from the 6th to 19th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 200 mg sulphamic acid/kg bw/day for the dams. At 600 mg sulphamic acid/kg bw/day several dams revealed signs of toxicity in form of behavioural changes and a reduction in the mean body weight and in food consumption in comparison to the control group. Necropsy revealed no changes for the dams of all treatment groups.

The no-observed-adverse effect level (NOAEL) for the fetal organism was also at 200 mg sulphamic acid/kg bw/day.

An increased number of resorptions, a slightly reduced fetal body weight and delayed ossifications for the os ischii, the sacral and caudal vertebral bodies and the metatarsalia were noted at the materno-toxic dose level of 600 mg sulphamic acid/kg bw/day. No dead fetuses, no malformations and no test item-related variations were noted at any of the tested dose levels. Thus, the substance is considered being non-embryotoxic.

In an OECD 443 study a daily administration of Sulfamic acid by oral gavage to Crl:WI (Wistar) rats at dose levels of 50, 150 and 500 mg/kg bw/day for a minimum of 17 weeks (Parental generation), followed by F1 generation pups selected for daily treatment by oral administration from weaning and terminated after the end of their treatment period (Cohort 1A), did not produce any macroscopic and microscopic test item-related findings.

There were no changes seen in the study that would suggest a generational effect of the test item on any of the evaluated parameters.

In conclusion, under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) values for Sulfamic acid was considered to be as follows:

• Based on test item effect on body weight, the NOAEL for systemic toxicity of parental generation: 500 mg/kg bw/day.


• Based on the effect on the reproductive parameters of parental animals (apparent lower male fertility at the High dose, and equivocal morphological sperm observations (restricted to the F0 generation), the NOAEL for reproductive toxicity of parental generation: 150 mg/kg bw/day.


• Due to lack of any test item effect on the body weight or food consumption of F1 generation, the NOAEL for physical development of F1 pups: 500 mg/kg bw/day.


• Due to lack of any test item effect on the sexual development of F1 generation, the NOAEL for sexual development of F1 pups: 500 mg/kg bw/day.


• No indication of any neurotoxic effect of the test item was indicated in the study.


• No indication of any immunotoxic effect of the test item was indicated in the study.


• No endocrine disruptor effect of the test item was indicated in the study.

 

Justification for classification or non-classification

Based on the outcome of an OECD414 and an OECD443 study, no reproductive toxicity was observed. Thus, the substance is not classified for reproductive toxicity according to CLP (Regulation EC No 127272008) or DSD (Directive 67/548/EEC).