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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
Deviations have been detailed in section "Pinciples of method if other than guideline".
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
For the repeated dose toxicity part, OECD TG 422 is in concordance with OECD TG 407 / EU B.7 except for the use of pregnant females, for which exposure duration (of female animals) is longer in OECD TG 422 compared to OECD TG 407 / EU B.7.
Principles of method if other than guideline:
Protocol deviations
An acclimatisation period of 20 days was allowed to animals prior to dosing (not 15 days as indicated in the Study Protocol). Litters from Dam nos. 17, 43 and 79 were observed for sex and presence on Day 14 (prior to necropsy) and not on Day 13 as indicated in the Study Protocol.
Equalisation of blood collection was carried out in the males only for coagulation.
These deviations are not considered to have compromised the purpose or conduct of the study. No other deviations occurred during the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium hydrogen m-sulphonatobenzoate
EC Number:
241-602-5
EC Name:
Sodium hydrogen m-sulphonatobenzoate
Cas Number:
17625-03-5
Molecular formula:
C7H6O5S.Na
IUPAC Name:
sodium 3-sulfobenzoate
Test material form:
solid: particulate/powder
Details on test material:
White crystalline powder.
Specific details on test material used for the study:
Alternative names: 3-Sulpho Benzoic Acid Mono Sodium Salt
SBA (3-Sodiosulfobenzoic Acid)
Sodium hydrogen m-sulphonatobenzoate
Label name: 3-Sodiosulfobenzoic Acid
EC number: 241-602-5
CAS number: 17625-03-5
Batch number: 170103
Date of expiry: 14 February 2019
Appearance: White crystalline powder
Storage conditions: Room temperature, protected from moisture

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
A total of 102 Hsd: Sprague Dawley SD rats (45 males and 57 virgin females), 6 to 7 weeks old and weighing 175 to 200 g for males and 151 to 175 g for females, from Charles River Italia S.p.A., Calco (Lecco), Italy.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal supply and acclimatisation
A total of 102 Hsd: Sprague Dawley SD rats (45 males and 57 virgin females), 6 to 7 weeks old and weighing 175 to 200 g for males and 151 to 175 g for females, were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy.
After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check were performed by a veterinarian.
An acclimatisation period of approximately 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations. Rats considered unsatisfactory were killed and where appropriate subjected to pathological examination. Unsatisfactory batches of animals were rejected before the start of treatment.

Animal husbandry
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray was held absorbent material which was inspected and changed daily.
After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5x26.6x18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.
Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at RTC.
Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were be recorded.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals recived the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
The vehicle was 0.5% aqueous solution of carboxymethylcellulose.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis has been performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (RTC Study no. A2772).
Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the homogeneity (in the case of suspensions) and concentration.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC. The software used for this activity was Empower® 2 Build No. 2154.
Duration of treatment / exposure:
Duration of treatment
Animals was dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy, for a minimum of 28 days.

Males
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice.

Females
Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes will be calculated according to the last recorded body weight.
Frequency of treatment:
Once a day, 7 days a week.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 male and 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
Each group was comprise 10 male and 10 female rats. Females were selected on the basis of pre-exposure oestrous cyclicity and animals that fail to exhibit regular cycles were not included in the groups.
The computerised system used in this study was the Xybion Path/Tox System, Version 4.2.2.

The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.
The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.
Positive control:
Not used.

Examinations

Observations and examinations performed and frequency:
Mortality
Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day.
Severely debilitated animals was be observed carefully. Animals judged to be in extremis were killed. A complete necropsy was performed in all cases.

Clinical signs
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

Clinical observations (Functional Observation Battery Tests)
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals.

Grip strength and sensory reactivity to stimuli
Once during the study, towards the end of treatment (Day 12 post partum for females with viable litters, where possible), 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements will be performed using a computer generated random order.

Motor activity assessment
Once during the study, towards the end of treatment (on Day 12 post partum for females with viable litters, where possible), 5 males and 5 females were randomly selected from each group and the motor activity measured (for approximately 5 minutes) by an automated activity recording device.

Body weight
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams and pups were also be weighed on Days 1, 4, 7 and 13 post partum. Dams were also be weighed just prior to necropsy.

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum, and on Day 7 and 13 post partum starting from Day 1 post partum.

Vaginal smears and oestrus cycle

Stock females
Oestrus cycle was monitored by vaginal smears in all stock females for at least 1 week before allocation in order to exclude from the study females with irregular cycle.

Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of allocation and during treatment period, up to positive identification of mating including not less than 2 weeks before the pairing.
Animals that exhibit irregular cycle were not included in the study.
The vaginal smear data was examined to determine the following:
a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also be taken from all females, before despatch to necropsy. No vaginal smears were taken from females sacrificed for humane reasons.

Mating
Mating was monogamous (one male to one female). Exceptions can arise in the case of occasional deaths of males. A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurs or 14 days have elapsed.

Parturition and gestation length
A parturition check was performed from Day 20 to Day 25 post coitum. Females which do not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of birth. The day of birth (when parturition is complete) is defined as Day 0 post partum.

Pups identification, weight and observation
As soon as possible, after parturition is considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1, 4, 7 and 13 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy.
Observation was performed once daily for all litters. After culling, all retained pups were sacrificed with the dams on Day 14 post partum.

Culling and pups selection for blood collection (serum hormone) at necropsy
On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter depending on the normal litter size. Partial adjustment (for example, 5 males and 3 females) is acceptable.
At least one culled male and one culled female were selected for serum hormone determination. If there are insufficient pups in a litter to had two surplus pups (ie: litters with 4 males and 4 females or less) 2 female pups were sacrificed for serum hormone determination in order to retain more male pups for nipple retention on Day 14 post partum. However, retained female pups in each litter should not be below 2. This means that for litters with 4 males and 2 females no pups were selected for hormone determination.

Anogenital distance (AGD)
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to a measure of pup size, preferably the cube root of body weight collected on Day 1 post partum.

Nipple count
On Day 13 post partum, nipple areolas were counted and recorded, if present, for all live male pups.
Sacrifice and pathology:
Clinical pathology investigations
- Haematology
- Coagulation tests
- Clinical chemistry
- Blood collection and thyroid hormone determination (T3, T4 and TSH)
- Bionalysis - Thyroid hormone determination (T3, T4 and TSH)

Euthanasia
Parental animals and those that have completed the scheduled test period were killed by exsanguination under isofluorane anaesthesia.
Animals sacrificed for humane reasons were killed with carbon dioxide.
Pups killed for humane reasons or those that have completed the scheduled test period (Day 4 or 14 post partum) were euthanized by intraperitoneal injection of Thiopenthal.
The males were killed after the mating of all females or after at least 28 days of treatment period.

Parental males
The females with live pups were killed on Day 14 post partum.

Parental females
The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after.

The females showing no evidence of copulation were killed after 25 days after the last day of the mating session.
The females which do not give birth were killed on post coitum Day 25 or shortly after.
Other examinations:
Blood samples
Blood samples were taken as detailed above.

Necropsy
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons or found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following:

Females
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities.

Pups
All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection.
All live pups sacrificed at Day 14 post partum were killed and examined for external abnormalities and sex confirmation by gonads inspection.
All pups with abnormalities were retained in 10% neutral buffered formalin.

Nipples retention
Nipples/areolae in male pups, where present were retained in 10% neutral buffered formalin. at Day 14 post partum

Organ weights
From all animals completing the scheduled test period, organs were dissected free of fat and weighed.

Parental animals
The ratios of organ weight to body weight was calculated for each animal.
At the discretion of the pathologist, organs may be weighed from animals dying or killed prior to terminal kill.
Thyroid was weighed from one male and female from each litter and preserved for possible histopathological examination. The thyroid weight was determined after fixation.

Tissues fixed and preserved
Samples of all the tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are detailed in the report. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
In the first instance the examination was restricted as detailed below:
a) Tissues specified in the report from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
b) Tissues specified in the report from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.
The examination could then be extended to include the remaining 5 males and 5 females (animals not evaluated for clinical pathology) of the control and the high dose group and/or to animals of the other dose groups, if treatment-related changes are observed in the high dose group.

Photomicrographs
Representative photomicrographs were taken of any treatment-related lesions.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n is more than 5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups wereassessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs occurred during the study.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in body weight and body weight gain were observed during the study in the treated animals, when compared to controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No changes of toxicological relevance were observed during the study in food consumption in either males or females.
Food efficiency:
no effects observed
Description (incidence and severity):
No changes of toxicological relevance were observed during the study in food consumption in either males or females.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No changes were recorded. The decrease of neutrophils recorded in females dosed at 100mg/kg/day was not dose related, therefore it was considered to be incidental.

Coagulation
Minimal decrease of prothrombin time was recorded in some females dosed at 300 and 1000 mg/kg/day (mean group value was 10% and 7% below controls, respectively). Due to the severity and the direction, this change was considered of no toxicological significance.

No changes of toxicological significance were recorded in haematological parameters and coagulation.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Triglycerides were increased in a number of females from all treated groups. Compared with mean control data, this finding was recorded in animal nos. X0760023 (100mg/kg/day, 3.1 fold), X0760051 (300mg/kg/day, 2.4 fold), X0760055 (300mg/kg/day, 3.7 fold), X0760061 (1000mg/kg/day, 3.7 fold) and X0760063 (1000mg/kg/day, 2.7 fold). Compared with controls, other sporadic changes of some biochemical parameters were recorded in a number of females, such as: increase of aspartate aminotransferase in animal no. X0760031 (100mg/kg/day, 3.5 fold), increase of bile acids in animal nos. X0760051 and X0760063 (300 and 1000mg/kg/day, 2.4 and 3.3 fold, respectively) and decrease of chloride in those receiving 1000mg/kg/day (5%). Due to the slight incidence and/or severity, these findings were considered to be of no toxicological relevance.

No adverse findings were observed by clinical chemistry examinations.

Thyroid hormone determination (Tables 12, 13; Appendices 11, 12)

Parental males
Compared with controls, thyroxine (T4) was increased in males dosed at 100 and 1000mg/kg/day (25% and 37%, respectively) and thyroid stimulating hormone (TSH) was decreased in those receiving 300 and 1000mg/kg/day (48% and 60%).

Pups - Day 14 post partum
No relevant changes were recorded.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Clinical observations (Functional Observation Battery Tests: removal from the home cage and open arena)
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Motor activity, Grip strength and sensory reactivity to stimuli
No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.
Statistically significantly higher landing footsplay was recorded in the females dosed at 100mg/kg/day, when compared to the control group. Due to the absence of dose-relation, it was considered of no neurotoxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant changes were observed on terminal body, absolute and relative organ weight of treated animals that completed the treatment period, when compared to the controls.
The statistically significant changes observed in absolute and relative liver weight of high dose male group animals sacrificed at term, could be comparable to control animals for histomorphology and therefore they are not considered toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animals that completed the treatment period and killed at termination did not show relevant macroscopic changes that could be considered treatment-related. The sporadic changes such as small thymus in one high dose female (no. X0760067) and pelvic dilatation observed respectively in three high dose males (nos. X0760062, X0760068 and X0760072) and two control females (nos. X0760001, X0760011) were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

No treatment-related changes were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic observations
No treatment-related changes were noted in animals sacrificed at the end of the treatment period. The apparently increased incidence of inflammatory reaction in the heart noted in the five randomly high dose treated males, after the extension of histopathological evaluation to the remaining control, low, mid- and high dose group males, was seen comparable to control animals.
The remaining lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology and/or physiological oestrous cyclic changes, commonly seen in this species and age under our experimental conditions.

Spermatogenic cycle
A detailed qualitative examination of the testes was performed on 5 randomly selected animals in control and high dose groups respectively. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as missing germcell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen.
Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952. The PAS-stained sections were used to identify the spermatogenic stages.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

No treatment-related changes were noted in animals sacrificed at the end of the treatment period. In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, no signs of treatment-related toxicity were observed following treatment with Sodium 3-sulfobenzoate, when administered to rats by oral route at dose levels of 100, 300 and 1000mg/kg/day, at any of the dose levels investigated. Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.
Executive summary:

The toxic effects on rats of both sexes after repeated dosing with Sodium 3-sulfobenzoate, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring were investigated according OECD 422. No signs of treatment-related toxicity were observed following treatment with Sodium 3-sulfobenzoate, when administered to rats by oral route at dose levels of 100, 300 and 1000mg/kg/day, at any of the dose levels investigated. Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

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