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Administrative data

Description of key information

In a GLP Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, conducted in accordance with OECD guideline 422 with male and femal Wister (Han) rats by oral gavage at doses of 0 (vehicle only), 100, 300, and 1000 mg/kg-bw/day, a NOAEL of 100 mg/kg was established based on adverse morphological changes in the urinary bladder and kidneys.

At 1000mg/kg, adverse histopathologicalchanges in the urinary bladder consisted ofurothelialhypertrophy in both sexes.This finding was considered adverse based on its hyperplastic nature. Adverse renal findings at this dose consisted of an increased incidence and/or severity of tubular basophilia and dilation in the kidneys of both sexes and renal tubular casts in males. For males, tubular dilation correlated with macroscopic pale discolouration and with an increase in kidney weight. Higher urea and creatinine were measured for both sexes and were also attributed to the observed renal lesions.This combination of findings was considered to be adverse at 1000 mg/kg for both sexes.

At 300mg/kg, adverse urothelial hypertrophy of the urinary bladder was present in males only.

Other non-adverse renal changes were recorded in females at 100 and 300mg/kg. Minimal degrees of tubular dilation in females at 100 and 300mg/kg were considered to be within normal limits. Tubular basophilia was recorded in females at 100 mg/kg at minimal degree and at slight degree in females at 300mg/kg. These occurred in the absence of other supportive adverse related renal changes at these dose levels and as such were not considered adverse.

At 1000 mg/kg, serum levels of T4 in F0 males were reduced by approximately 50% compared to control values. This marked reduction of total T4 occurred in the absence of corroborating changes in either organ weight or morphology of the thyroid gland. Given the magnitude of the observed decrease in total T4, it was considered to represent a compound-related effect. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2017 - 12 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification: Sodium 2,4,5-trichlorobenzenesulphonate
CAS number: 53423-65-7
Acronym: STB-FR
Molecular formula: C6H2Cl3O3S.Na
Molecular weight: 283.494
Appearance: White powder
pH: 6.9 (5% aqueous slurry)
Batch: 1708-02
Purity/Composition: >99%
Stable under storage conditions until: 31 December 2018 (expiry date)
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The test laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
Age of Males (on Treatment Day 1): Approximately 10 - 12 weeks old
Age of Females (on Treatment Day 1): Approximately 12 - 14 weeks old
Weight of Males (on Treatment Day 1): 271 - 299 grams
Weight of Females (on Treatment Day 1): 198 - 248 grams
Health Status: All males and females were inspected prior to dosing and found to be healthy
Animal Identification: Earmark and Tattoo; Reserve females were randomly selected and numbered R1 through R8 with indelible marker. Pups were randomized and identified by injection of Indian Ink and, as needed tatoo on the feet.
Acclimitization: 7 days prior to start of the pretest period (females) or 6 days before the commencement of dosing (males)
Housing: Appropriate depending on stage of the study. Refer to full study report.
Env. Conditions: Temp: 20 - 22 deg Celsius; Relative Humidity: 43 - 57%; Lighting: 12-hour light/dark cycle; Air Changes: >=10/hour with 100% fresh air in the room.
Food: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum
Water: Municipal tap water was freely available to each animal via water bottles
Nesting Material: Paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom)
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Frequecy: Once during week 1 of dosing
Dose Groups Tested: Concentration: All Groups; Homogeneity: Groups 2 and 4
Method: LC-DAD (Method Validated by Analytical Biochemical Laboratory B.V. (ABL)
Stability of Test Substance in Vehicle: Verified to be stable during method development
Duration of treatment / exposure:
Males: 29 days up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
Females that failed to deliver: 41-51 days
Females that delivered: 50 - 62 days; i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Frequency of treatment:
Once daily, 7 days per week.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle only
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose Level Selection: Based on the results of a 10-day dose range finder with oral gavage administration in an attempt to produce graded responses to the test item.
Dose Volume: 5 ml/kg
Positive control:
No
Observations and examinations performed and frequency:
F0 Generation:
Mortality/ moribundity: Twice daily, in the morning and at the end of the working day
Clinical signs: Once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy
Functional observations (for 5 selected animals/sex/group; hearing, pupillary reflex, static righting reflex, strength, locomotor activity): Males: week 4 of treatment; Females: during last week of lactation (PND 6 - 13).
Body weight: Weekly
Food consumption: Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal day (PND) 1, 4, 7, and 13
Estrous cycle determination: All femails daily begining 13 days prior to treatment, the first 14 days of treatment, and during mating until evidence of copulation was observed.
Clinical pathology (for 5 selected animals/sex/group): Blood was collected once on the day of scheduled necropsy and included typical hematology and clinical chemistry parameters, and measurement of thyroid hormone T4 (F0-males).

F1 Generation:
Mortality/ moribundity: Daily
Clinical signs: At least once daily
Body wieght: Live pups were weighed individually on PND 1, 4, 7 and 13
Sex: Sex was externally determined for all pups on PND 1 and 4
Anogenital distance (AGD): Measured for all live pups on PND 1
Areola/Nipple Retention: All male pups in each litter were examined for the number of areola/nipples on PND 13
Culling: To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup).
Clinical pathology: Blood of F1-animals was collected on PND 4 and PND 14-16, if possible in included typical hematology and clinical chemistry parameters, and measurement of thyroid hormone T4 (2 pups per litter)
Sacrifice and pathology:
All animals were subjected to a full post mortem examination.

Organ Weights (paired together, as appropriate: brain, cervix, epididymis, adrenal gland, coagulation gland, parathyroid gland, prostate gland, seminal vesicle gland, thyroid gland, heart, kidney, liver, ovaries, spleen, testes, thymus, and uterus.

Tissue Collection/Preservation/Examination: Animal ID, aorta artery, nasopharynx body cavity, bone marrow, femur, sternum, brain (seven levels), cervic, epididymis, esophagus, eye, adrenal gland, coagulation gland, harderian gland, lacrimal glan, mammary gland, parathyroid gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, cecum, colon, rectum, larynx, liver, lung, mandibular and meenteric lymph nodes, skelatal muscle, optic nerve, sciatic nerve, ovaries, pancreas, skin, duoenum, ileum, jejunum, spinal cord, spleen, stomach, testes, thymus, tongue, tracea, urinary bladder, uterus. amd vagina.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female no. 52 (100 mg/kg) died at blood sampling before necropsy on Lactation Day 14. There were no test item-related macroscopic or microscopic findings in the organs examined for this animal. This accidental death was attributed to the blood sampling procedure, and as such not considered to be related to treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slight, non-adverse, changes in body weight were noted for males and females at 1000 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Slight, non-adverse, changes in food intake were noted for males and females at 1000 mg/kg
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Higher alanine aminotransferase activity (ALAT) in males at 300 and 1000 mg/kg (1.5x higher than control values; means outside historical control range).
- Higher aspartate aminotransferase activity (ASAT) in males at 300 and 1000 mg/kg (1.3x and 1.5x higher than control values, respectively; means within historical control range).
- Higher urea in males and females at 1000 mg/kg (2.0x and 1.2x of control, respectively; mean outside historical control range).
- Higher creatinine in males at 1000 mg/kg (1.7x higher than control; mean outside historical control range).
- Lower potassium in males at 1000 mg/kg (0.9x of control; mean outside historical control range).
- Lower mean total bilirubin in females at 1000 mg/kg (0.8x of control; mean within historical control range).
- Higher cholesterol in females at 1000 mg/kg (1.3x of control; mean within historical control range).
- Lower serum levels of T4 in F0 males at 1000 mg/kg. Levels were 0.5x lower than control values. For three males at 1000 mg/kg (nos. 31, 33 and 36), the T4 value was below the detection limit of 1.00 ug/dL.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There was an apparent test item-related decrease in thymus weight in males at 100, 300 and 1000 mg/kg and in females at 300 and 1000 mg/kg, but this was only significant in males treated at 1000 mg/kg/day (absolute). In addition, there was a test item-related increase in kidney weight in males treated at 1000 mg/kg/day (absolute and relative to body weight).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were present in males treated at 1000 mg/kg/day in the kidneys as pale discolouration in 5/10 rats and in the thymus as reduced in size in 1/10 rat.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with STB-FR were noted in the urinary bladder, kidneys, thymus and stomach of both males and females. Urothelial hypertrophy of the urinary bladder was present in males at 300 and 1000 mg/kg (up to moderate degree) and in females at 1000 mg/kg (up to slight degree). An increased incidence and/or severity of tubular basophilia in the kidneys was present in males at 1000 mg/kg (up to moderate degree) and in females at 300 and 1000 mg/kg (up to slight degree). Renal casts were present at increased incidence and severity in males at 1000 mg/kg (up to slight degree). Renal tubular dilation was present at increased incidence and severity in males and females at 1000 mg/kg (up to marked and slight degree, respectively). For males, tubular dilation correlated with the macroscopic pale discolouration and with the increase in kidneys weight. Lymphoid depletion in the thymus was present at minimal degree in females at 300 and 1000 mg/kg and in males at 1000 mg/kg. For males, this correlated with the macroscopic reduction in size and with the decrease in thymus weight. Squamous cell hyperplasia of the limiting ridge of the stomach was present at minimal degree in males and females at 1000 mg/kg.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Summary of Test Item-Related Microscopic Findings Urinary Bladder, Kidneys, Thymus and Stomach

 

Males

Females

Dose

0

100

300

1000

0

100

300

1000

URINARY BLADDER

5

5

5

5

5

5

5

5

Hypertrophy urothelium

 

 

 

 

 

 

 

 

 - Minimal

---

---

2

2

---

---

---

2

 - Slight

---

---

1

2

---

---

---

2

 - Moderate

---

---

---

1

---

---

---

---

KIDNEY

5

5

5

8

5

5

5

5

Basophilia tubule

 

 

 

 

 

 

 

 

 - Minimal

1

3

4

1

---

1

---

1

 - Slight

---

---

---

4

---

---

2

1

 - Moderate

---

---

---

2

---

---

---

---

Casts

 

 

 

 

 

 

 

 

 - Minimal

---

---

---

1

1

---

---

1

 - Slight

---

---

---

6

---

---

---

---

Dilation tubule

 

 

 

 

 

 

 

 

 - Minimal

---

---

1

---

---

1

1

1

 - Slight

---

---

---

3

---

---

---

2

 - Moderate

---

---

---

3

---

---

---

---

 - Marked

---

---

---

1

---

---

---

---

THYMUS

5

5

5

5

5

5

5

5

Depletion lymphoid

 

 

 

 

 

 

 

 

 - Minimal

---

---

---

2

---

---

2

1

STOMACH

6

5

5

5

5

5

5

5

Hyperplasia squamous cell limiting ridge

 

 

 

 

 

 

 

 

 - Minimal

---

---

---

4

---

---

---

2

Conclusions:
A NOAEL of 100 mg/kg was established based on adverse morphological changes in the urinary bladder and kidneys, which were minimal to slight in males only at 300 mg/kg. The relevance of these findings to humans is unknown. Given the adverse effects observed, incidence, dose level, study duration, and unknown relevance to humans, the GHS criteria for classification under specific target organ toxicity (STOT) have not been met.
Executive summary:

In this GLP Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, conducted in accordance with OECD guideline 422 with male and femal Wister (Han) rats by oral gavage at doses of 0 (vehicle only), 100, 300, and 1000 mg/kg-bw/day, a NOAEL of 100 mg/kg was established based on adverse morphological changes in the urinary bladder and kidneys.

At 1000 mg/kg, adverse histopathological changes in the urinary bladder consisted of urothelial hypertrophy in both sexes.This finding was considered adverse based on its hyperplastic nature. Adverse renal findings at this dose consisted of an increased incidence and/or severity of tubular basophilia and dilation in the kidneys of both sexes and renal tubular casts in males. For males, tubular dilation correlated with macroscopic pale discolouration and with an increase in kidney weight. Higher urea and creatinine were measured for both sexes and were also attributed to the observed renal lesions.This combination of findings was considered to be adverse at 1000 mg/kg for both sexes.

At 300 mg/kg, adverse urothelial hypertrophy of the urinary bladder was present in males only.

Other non-adverse renal changes were recorded in females at 100 and 300 mg/kg. Minimal degrees of tubular dilation in females at 100 and 300 mg/kg were considered to be within normal limits. Tubular basophilia was recorded in females at 100 mg/kg at minimal degree and at slight degree in females at 300mg/kg. These occurred in the absence of other supportive adverse related renal changes at these dose levels and as such were not considered adverse.

At 1000 mg/kg, serum levels of T4 in F0 males were reduced by approximately 50% compared to control values. This marked reduction of total T4 occurred in the absence of corroborating changes in either organ weight or morphology of the thyroid gland. Given the magnitude of the observed decrease in total T4, it was considered to represent a compound- related effect. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.

Given the adverse effects observed, incidence, dose levels, study duration, unknown relevance to humand, and the resulting NOAEL of 100 mg/kg, the GHS criteria for classification under specific target organ toxicity (STOT) were not met.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Adequate for the tonnage band being registered.
System:
urinary
Organ:
bladder
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The observed effects are of unknown relevance to humans but is suspected as not being specifically relevant given that the effects were only seen at the highest dose tested or at a lower dose in male rats only.

Additional information

Justification for classification or non-classification

Given the adverse effects observed, incidence, dose levels, study duration, unknown relevance to humans, and the resulting NOAEL of 100 mg/kg, the GHS criteria for classification under specific target organ toxicity repeated exposure (STOT-RE) were not met.