Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
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)

Test animals

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)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
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.
Doses / concentrationsopen allclose all
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

Examinations

Parental animals: Observations and examinations:
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).
Oestrous cyclicity (parental animals):
All femails daily begining 13 days prior to treatment, the first 14 days of treatment, and during mating until evidence of copulation was observed.
Sperm parameters (parental animals):
For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
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)
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
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.
Reproductive indices:
Mating Index (%): # of females mated / # of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating.
Fertility Index (%): # of pregnant females / # of females mated x 100
Gestation Index (%): # of females with living pups on Day 1 / # of pregnant females x 100
Duration of Gestation: Number of days between confirmation of mating and the beginning of parturition.
Post-implantation survival index (%): Total # of offspring born / Total number of uterine implantation sites x 100
Live birth index (%): # of live offspring Day 1 after littering / Total number of offspring born x 100
Percentage live males at First Litter Check (%): # of live male pups at First Litter Check / # of live pups at First Litter Check x 100
Percentage live females at First Litter Check (%): # of live female pups at First Litter Check / # of live pups at First Litter Check x 100
Offspring viability indices:
Viability Index (%): # of live offspring on Day 4 before culling / # of live offspring on Day 1 after littering x 100
Lactation Index (%) # of live offspring on Day 13 after littering / # of live offspring on Day 4 (after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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
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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
clinical biochemistry
gross pathology

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
A NOAEL of >= 1000 mg/kg was established based on the absence of any adverse reproductive effects at the highest dose tested. Given these results, the substance does not meet the GHS criteria for classification under the criteria for reproductive toxicity.
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 female Wister (Han) rats by oral gavage at doses of 0 (vehicle only), 100, 300, and 1000 mg/kg-bw/day, a NOAEL of 1000 mg/kg-bw/day was established for reproductive toxicity based on the absence of any adverse reproductive effects at the highest dose tested. Given these results, the substance does not meet the GHS criteria for classification under the criteria for reproductive toxicity.