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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 FEB 2021- 19 OCT 2021 (experimental phase: 09 MAY 2021 - 26 JUN 2021)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000.
Deviations:
yes
Remarks:
Mating period was prolonged by a day to ensure a successful mating for two pairs at 100 mg/kg bw/day.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 July 2016.
Deviations:
yes
Remarks:
Mating period was prolonged by a day to ensure a successful mating for two pairs at 100 mg/kg bw/day.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
5-sulphosalicylic acid
EC Number:
202-555-6
EC Name:
5-sulphosalicylic acid
Cas Number:
97-05-2
Molecular formula:
C7H6O6S
IUPAC Name:
2-hydroxy-5-sulfobenzoic acid
Test material form:
solid: crystalline
Details on test material:
Batch number 1: 1907015
Batch number 2: 2005014
CAS number: 5965-83-3
EC number 202-555-6
Molecular formula: C7H6O6Sx2H2O
Appearance: White to off white crystals
Assay: 101.9 % – batch 1
102.2 % – batch 2
Manufacturing date. July 15, 2019 – batch 1
May 01, 2021 – batch 2
Expiry date: July 31, 2021 – batch 1
April 30, 2022 – batch 2
Storage conditions: At room temperature (15 – 25°C); protected from light
Safety precautions: According to safety data sheet

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han:WIST of Wistar origin
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 82 –89 days
- Weight at study initiation: 348 – 402 g for male animals, 211 – 261 g for female animals
- Housing:
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Mated females: individually
Males after mating: 2 animals / cage
- Diet (e.g. ad libitum): ad libitum. ssniff® SM R/M-Z+H complete diet for rats and mice. Food was changed at weekly intervals.
- Water (e.g. ad libitum): ad libitum. changed daily
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): Above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: one female and one male of the same dose group (1:1 mating) placed in a single cage.
- Length of cohabitation: Females remained with the same male until copulation occurred.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.
- Any other deviations from standard protocol: Mating period was prolonged by a day to ensure a successful mating for two pairs at 100 mg/kg bw/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five aliquots of 5 mL of each formulation and five aliquots of control substance (vehicle) were taken two times and were analyzed.
Date of sampling: March 17 and April 22, 2021
Date of analysis: March 17 and April 23, 2021
Concentration of the test item in the dosing formulations varied between the range of 98.3 – 105 % in comparison to the nominal values.

5-Sulfosalicylic acid was quantified in dosing formulations by high performance liquid chromatography coupled with UV detection in the concentration range of 0.05 – 0.25 mg/mL.
The dosing formulations were diluted 100×, 200× or 1000× for the analysis.
Linear range: 0.05 - 0.25 mg/mL
Limit of Quantification 0.05 mg/mL
Recovery: 93.2 % (1 mg/mL); 95.3 % (150 mg/mL)
Selectivity: No interfering component was detected in the blank sample (ultrapure water) and in the blank formulation (Aqua purificata)
Duration of treatment / exposure:
Male: From Day 0 up to Day 49, (March 09 – April 27, 2021), daily up to the day before the necropsy (during the pre-mating, mating and post-mating periods).
Female: From Day 0 up to Day 63, (March 09 – May 11, 2021), daily up to the day before the necropsy (during the pre-mating, mating, gestation and lactation periods).
Frequency of treatment:
administered orally (by gavage) once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
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:
12 animals per sex per dose
12 animals for control group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary dose range finding study with 5-Sulfosalicylic Acid in rats (Study no. 968-400-5401).
- Rationale for animal assignment (if not random): Selected rats were distributed by randomization according to stratification by body weight so that there was no statistically significant difference among group body weight means within a sex.
- Fasting period before blood sampling for clinical biochemistry: Animals were food deprived for approximately 16 hours (overnight) prior to blood collection.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Day 0, (March 09, 2021), then weekly and on the day before necropsy;

BODY WEIGHT: Yes
- Time schedule for examinations:
Male: From Day 0 up to Day 48, (March 09 – April 26, 2021), weekly, (during the pre-mating, mating and post-mating periods)
Female: From Day 0, (March 09), weekly prior to and during the mating period. On gestational days 0, 7, 10, 14 and 21. On post-partum days 0, 4 and 13
Body weight of parental animals selected for organ weighing were determined on the day of necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Male: From Day 0 up to Day 48, (March 09 – April 26, 2021), weekly prior to and after the mating period.
Female: From Day 0, (March 09, 2021), weekly prior to and after the mating period. Also on gestational days 0, 7, 14, and 21 and on post-partum days 0, 4 and 13

Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears from each animal being considered for study each day for two weeks before the treatment started. Estrous cycle was evaluated and considered at randomization.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations: The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), Male copulatory index, Male fertility index; weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined at the time of termination.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 2-7 pups/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Litter weight on post-natal days 0, 4 and 13, Mean body weight gain per litter between post-natal days 0-4, 4-13 and for overall post-natal days, Number of live births per litter, and number of viable pups per litter on post-natal days 0, 4 and 13, Survival Index of pups on post-natal day 13, Sex ratio % (on post-natal days 0 and 13), Normalized anogenital distance, Number of nipples/areolae in male pups, FT3, FT4 and TSH levels (on post-partum day 13)

GROSS EXAMINATION OF DEAD PUPS: yes, necroscopy by macroscopic examination on the day when they were found dead. On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups diesd intrauterine from pups died after the birth.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Male animals were dosed for 50 days (14 days pre-mating and 1-15 days mating, plus 21-35 days of post-mating period; until the necessary number of pregnant female animals was evident); then they were sacrificed on Day 50.
- Maternal animals: Females were dosed for 14 days pre-mating, through 1-15 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice on Days 50 -55 or on Day 64.

GROSS NECROPSY
All animals were subjected to a full detailed gross necropsy

HISTOPATHOLOGY / ORGAN WEIGHTS
The body weight, brain weight and weight of the testes and epididymides and prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.
Postmortem examinations (offspring):
SACRIFICE
F1 offspring were observed for clinical signs, litter weight, body weight, anogenital distance and nipple retention. Blood samples for serum FT3, FT4 and TSH assessment were pooled from 2-7 pups per litter on post-natal day 4 (in litters with at least 10 pups) and from 3-7 pups per litter on post-natal day 13. Remaining pups were euthanized on post-natal day 13 or shortly thereafter.

GROSS NECROPSY
on post-natal day 13 or shortly thereafter

HISTOPATHOLOGY / ORGAN WEIGTHS
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (1000 mg/kg bw/day) groups. Additionally, kidneys, liver with diaphragm, skin, stomach, seminal vesicles and uterus showing macroscopic findings at necropsy were processed and evaluated histologically in some animals and offspring
Statistics:
The statistical evaluation of appropriate data (marked above) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Reproductive indices:
Copulatory index: Measure of animals’ ability to mate
Fertility index: Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Gestation index: Measure of pregnancy that provides at least one live pup
Offspring viability indices:
Post-implantation mortality (intrauterine mortality)
Post-natal mortality
Survival Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Alopecia, scars and porphyrin around the eye – as species specific findings of this species and strain of rat with similar age – was noted with low incidence.
Dermal irritation (if dermal study):
not specified
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Statistical significance was only detected at the slightly higher mean body weight gain in male animals at 100 mg/kg bw/day between Days 27 and 34.
This minor change was were considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Statistical significance was noted for the slightly higher mean daily food consumption in male animals at 300 mg/kg bw/day comparing to their control between Days 27 and 34 and between Days 34 and 41.
This minor difference was considered to be indicative of biological variation and not related to test item administration.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Statistically significant difference with respect their control was detected at the higher mean platelet count (PLT) in male animals at 100 and 300 mg/kg bw/day independently from doses. In the female animals at 100 and 300 mg/kg bw/day, statistical significance with respect to the control was observed at the slightly lower mean percentage of monocytes (MONO), at the lower mean corpuscular hemoglobin content (MCH) and at the lower mean corpuscular volume (MCV), when compared to the control. At 1000 mg/kg bw/day, the mean corpuscular hemoglobin content, mean corpuscular volume and prothrombin time (PT) were lower than in the control group in the female animals. All these changes were considered to be variations and toxicologically not relevant due to the minor degree or the lack of dose dependency (PLT, MONO, MVH, MCHC and PT). Individual values were within the historical control ranges except for PLT of one male animal at 100 mg/kg bw/day. Therefore, these findings were judged to be accidental.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Statistical significance noted for the slightly lower mean glucose (GLUC) concentration in the male animals at 1000 mg/kg bw/day was considered to be not related to the treatment or test item. The individual values of glucose level were well within the historical control range. In the female animal, all examined clinical chemistry parameters were comparable with the control at 100, 300 and 1000 mg/kg bw/day.
Endocrine findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the selected male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the male animals, right or left sided pyelectasia was seen in the control group (1/12), in 100 and 1000 mg/kg bw/day groups (2/12 and 1/12, respectively). In one male animal at 300 mg/kg bw/day, the right-side seminal vesicle was smaller than normal and empty, i.e., did not contain seminal fluid. Sign of diarrhea (face contaminated hairs around the anus) was observed in several male animals (6/12) at 1000 mg/kg bw/day on the day of necropsy. These findings were considered to be toxicologically not relevant in the present study.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Histopathological investigations did not reveal test item related lesions in the organs or tissues of animals selected for toxicity examinations at 1000 mg/kg bw/day (male and female).
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
A test item influence on the estrous cycle was not detected at any dose level (100, 300 and 1000 mg/kg bw/day).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of epididymides, prostate, seminal vesicles and coagulating glands was normal in all cases as well except for one male animal at 300 mg/kg bw/day. In accordance with macroscopic observation, decreased amount of secrete was observed in the seminal vesicle of this animal (1/12). However, this lesion in the seminal vesicle had no influence on the fertility of this animal.
Reproductive performance:
no effects observed
Description (incidence and severity):
Statistical significance with respect to the control was detected at the slightly lower fertility index (lower percentage of male animals fertilizing females) at 1000 mg/kg bw/day as mating of two pairs was infertile.
Similarly, the fertility index of female animals (percentage of pregnant females) was slightly lower than in the control group at 1000 mg/kg bw/day. Values met well the historical control.
Test item effect was not assumed because data met well historical control value both in male and female animals and in the lack of related findings in the reproductive parameters.

Details on results (P0)

Under the conditions of the present study, 5-Sulfosalicylic Acid administered at 100, 300 and 1000 mg/kg bw/day oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) or systemic toxicity in parental male and female Han:WIST rats as far as investigated in this study.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general systemic toxicity
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

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical signs were observed with variable incidence in the control, 100, 300 or 1000 mg/kg bw/day groups - cold body temperature, not suckled, smaller than normal, hematoma – independently from doses. The percentage of pups with clinical signs in the control group exceeded that in 100, 300 and 1000 mg/kg bw/day groups. These signs are commonly observed in offspring of not treated dams of this strain and showed no dose dependency in this study. Therefore, these observations were considered to have no toxicological relevance.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality. There were no significant differences between the control and test item treated (100, 300 or 1000 mg/kg bw/day) groups in the survival indices.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean litter weights and mean pup weights as well as the mean litter weight gains and mean pup weight gains were mostly similar in the control and in all test item treated groups (100, 300 and 1000 mg/kg bw/day) on post-natal days 0, 4 and 13. The mean pup weight (PND 4) and mean pup weight gain (PND 0 - 4) were slightly lowered comparing to the control at 100 mg/kg bw/day on post-natal day 4.
Evaluating the body weight separately for male and female offspring, statistical significance was observed at the slightly lower mean body weight of male and female pups at 100 mg/kg bw/day comparing to their control on post-natal day 4.
These findings were considered being accidental and to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Statistical significance was observed at the slightly longer mean absolute anogenital distance in male offspring at 1000 mg/kg bw/day. In the female offspring, the mean normalized anogenital distance was slightly higher than in the control at 100 mg/kg bw/day. These minor changes in male and female offspring were considered as biological variations and were considered to be independent from dam’s treatment. All values met well the historical control ranges.In the male offspring, the mean absolute and normalized anogenital distance were comparable in the control and 100 and 300 mg/kg bw/day groups.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Nipples/areoles were not visible in any of the examined male offspring in the control or 100, 300 or 1000 mg/kg bw/day groups on post-natal day 13.
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
Histological examination of kidneys in PND13-16 offspring did not reveal degenerative, inflammatory or other histological (fibrotic etc.) lesion. The incidence of renal change was comparable in the offspring of control and high dose treated dams, therefore pyelectasia was considered as a common finding in laboratory rats without toxicological significance.
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 100, 300 and 1000 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows: NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effect levels (F1)

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:
other: no effects in highest dose group

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on the observations, the No. Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/ female parental rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/ female parental rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day

Executive summary:

Toxicity to reproduction study was performed according to the OECD Guideline 422 under GLP compliance. Under the conditions of the present study, the test sample administered at 100, 300 and 1000 mg/kg bw/day oral gavage did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) or systemic toxicity in parental male and female Han:WIST rats as far as investigated in this study. The development of the F1 offspring was not impaired from birth up to post-natal day 13 as far as investigated in this study after repeated oral administration of dams at 100, 300 or 1000 mg/kg bw/day.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male/ female parental rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of male/ female parental rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day