2-phenyl-1H-benzimidazole-5-sulphonic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The test substance had been investigated for androgen and estrogen binding effects and was found absent of having such effects. Also an in-vivo uterotrophic screening assay following repeat subcutaneous administration did not reveal endocrine effects.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Reference 2

Endpoint:

two-generation reproductive toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Neither embryotoxic nor teratogenic effects were observed in a rat developmental toxicity study dosed up to 1000 mg/kgbw during gestational days 6 - 15.

 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 19 - Dec 10, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission of the European Communities - EEC Directive 79-831, Annex V, Part B, Toxicological Methods of Annex VIII, Teratogenicity Test. Official Journal of the European Communities L 133/24-26, May 30, 1988.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Versuchstierzucht GmbH in 33178 Borchen
- Age at study initiation: appr. 9 weeks
- Weight at study initiation: 166 (154 - 178) g
- Fasting period before study: no
- Housing: individually under conventional conditions in Makrolon cages (type III) on softwood granulate specially manufactured for housing of animals
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23 °C
- Humidity (%): 47 - 65 %
- Photoperiod (hrs dark / hrs light): 12 hours /12 hours

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
- Concentration in vehicle: 20 %
- Amount of vehicle (if gavage): 100 mL
- Lot/batch no. (if required): --
- Purity: distilled

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused]
- M/F ratio per cage: 4 f / 1 m
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

treatment on 10 consecutive days from the 6th to the 15th day post conception

Frequency of treatment:

daily

Duration of test:

20 days

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 3, and 6 p.c., and then daily until the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was determined once a week (on days 6, 10, 15, and 20) by weighing the unconsumed food.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Water consumption was determined on days 3, 6, 9, 12, 15, 18, and 20 by weighing the water not consumed.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
The fetuses were delivered by Cesarean section on the 20th day p.c. and were examined for macroscopic malformations. About 2/3 of them were examined for skeletal and 1/3 for organ malformations.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: 1/3
- Skeletal examinations: Yes: 2/3

Statistics:

Standard statistical methods have been applied for data processing.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption in group 2 was increased during the treatment period and further until day 18 p.c.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

not examined

Total litter losses by resorption:

not examined

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Complete, early, and late resorptions in group 2 were within the spontaneous range.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects: no effects

Details on maternal toxic effects:
All dams were clinically normal and none of the rats died.
Food consumption was normal, but water consumption in group 2 was increased during the treatment period and further until day 18 p.c.
Body weight was not negatively affected by treatment.
23 rats in group 1 (control) and 24 rats in group 2 (treatment) became pregnant.

The numbers of corpora lutea and live fetuses in group 2 corresponded to those in group 1. The number of implantations was reduced. Since implantation had been completed at the beginning of treatment, this finding is not substance related and, thus, irrelevant.

Complete, early, and late resorptions in group 2 were within the spontaneous range. No dead fetuses were found.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At external examination one fetus with anemia was observed in group 1 (control).

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
Fetal weights were normal.
No runts were seen.
Sex distribution was normal.
At external examination one fetus with anemia was observed in group 1 (control).
No other abnormal findings were seen at external examination, evisceration, skeleton staining and after transverse section.
The skeletal examinations gave no indication of damage.

Key result

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Key result

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Apart from the increased water consumption, Phenylbenzimidazole sulfonic acid (Na-salt) did not reveal maternally toxic effects and was neither embryotoxic nor teratogenic at the limit dose of 1000 mg/kg bw/day.

Executive summary:

Study design

The teratogenic potential of Phenylbenzimidazole sulfonic acid (Na-salt) was investigated in Wistar rats after oral administration in a limit test. The test item was administered orally by gavage to 25 mated Wistar rats daily from day 6 through to day 15 post coitum at the limit dose level of 1000 mg/kg bw/day. A control group of 25 animals was dosed with the vehicle alone (water). All females were sacrificed on day 20 post coitum and the fetuses were removed by Caesarean section and examined for macroscopic malformations. About 2/3 of them were examined for skeletal and 1/3 for organ malformations.

Results

All females survived until scheduled necropsy. No signs of discomfort or clinical symptoms were observed. Mean food consumption and mean body weight was not affected by treatment with the test item in the dose group. In the dose group water consumption was increased during the treatment period and further until day 18 post coitum. 23 rat in the control group and 24 rats in the dose group became pregnant. The numbers of corpora lutea and live fetuses in the dose group corresponded to those in the control animals. Complete, early and late resorptions in the dose group were within the spontaneous range. No dead fetuses were recognized.
No effects on fetal body weights were noted. No test-item-related effects on fetal sex ratio were noted in the dose group. During the external examination of the fetuses, no test item-related abnormal findings were noted. No test item-related abnormalities were noted during visceral examination of fetuses. No abnormalities, which were considered to be test item-related, were noted during examination of fetal skeleton.

Conclusion

Apart from the increased water consumption, Phenylbenzimidazole sulfonic acid (Na-salt) did not reveal maternally toxic effects and was neither embryotoxic nor teratogenic at the limit dose of 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The teratogenic potential of Phenylbenzimidazole sulfonic acid (Na-salt) was investigated in Wistar rats after oral administration in a limit test. The test item was administered orally by gavage to 25 mated Wistar rats daily from day 6 through to day 15 post coitum at the limit dose level of 1000 mg/kg bw/day. A control group of 25 animals was dosed with the vehicle alone (water). All females were sacrificed on day 20 post coitum and the fetuses were removed by Caesarean section and examined for macroscopic malformations. About 2/3 of them were examined for skeletal and 1/3 for organ malformations.

All females survived until scheduled necropsy. No signs of discomfort or clinical symptoms were observed. Mean food consumption and mean body weight was not affected by treatment with the test item in the dose group. In the dose group water consumption was increased during the treatment period and further until day 18 post coitum. 23 rats in the control group and 24 rats in the dose group became pregnant. The numbers of corpora lutea and live fetuses in the dose group corresponded to those in the control animals. Complete, early and late resorptions in the dose group were within the spontaneous range. No dead fetuses were recognized and no effects on fetal body weights were noted. No test-item-related effects on fetal sex ratio were noted in the dose group. During the external examination of the fetuses, no test item-related abnormal findings were noted. No test item-related abnormalities were noted during visceral examination of fetuses. No abnormalities, which were considered to be test item-related, were noted during examination of fetal skeleton. Apart from the increased water consumption, Phenylbenzimidazole sulfonic acid (Na-salt) did not reveal maternally toxic effects and was neither embryotoxic nor teratogenic at the limit dose of 1000 mg/kg bw/day. The substance did not show any notable effect on fertility or developmental toxicity in a 90d oral repeat toxicity study, although dosed up to 1000 mg/kg bw/d (NOAEL was 1000 mg/kg bw/d) and in addition the oral developmental toxicity study did not show embryotoxicity, or teratogenicity (NOEL >1000 mg/kg bw/d). Thus, in line with the criteria in REACH, Annex IX 8.7.3, column 1 there is no need at this tonnage level to further investigate developmental toxicity by a multi-generation study and such a study is waived.

Toxicity to reproduction: other studies

Link to relevant study records

Reference

Reference 1

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: scientifically valid method was used and study is adequately reported

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of this in vitro investigation was to evaluate a potential affinity of test article sodium salt for the androgen receptor (AR). The experimental system employed was capable of detecting strongly and weakly binding compounds such as dihydrotestosterone and androstenedione.
ln principle, the method described by Boesel and Shain (A rapid, specific protocol for determination of available androgen receptor sites in unfractioned rat ventral prostate cytosol preparations. Biochem Biophys Res Comm 61: 1004-1011, 1974) was applied, however, a rat recombinant fusion protein containing both the hinge region and ligand binding domain of the AR served as receptor source, whereas radiolabeled methyltrienolone was used as receptor ligand according to Kelce et al. (Kelce WR, Monosson E, Gamcsik MP, Laws SC, Gray LE jr., Environmental hormone disruptors: Evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites. Toxicol Appl Pharmacol 126: 276-285, 1994).

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Details on test animals or test system and environmental conditions:

Biological material:
Androgen receptor (AR): rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain of the AR (PanVera, Madison, Wl, USA).
Equipment:
Labofuge A (Heraeus, Osterode, FRG)
Liquid scintillation counter 1900 TR (Canberra-Packard, Frankfurt, FRG)
Liquid scintillation counter 1450 Microbeta™ Trilux (Wallac, Freiburg, FRG)
Chemicals:
Tris(hydroxymethyl)-aminomethane (Tris) p.a. (E. Merck, Darmstadt, FRG)
Sodium chloride (KMF, St. Augustin, FRG)
Dithiothreitol (DTT ) (Sigma, Deisenhofen, FRG)
Glycerol anhydrous pure (E. Merck, Darmstadt, FRG)
Human y-globulin 96 - 99 % (Sigma, Deisenhofen, FRG)
Dimethylsulfoxid p.a. (DMSO) (E. Merck, Darmstadt, FRG)
Ethanol p.a. (E. Merck, Darmstadt, FRG)
Ultima Gold™ scintillation cocktail (Canberra-Packard, Frankfurt, FRG)
Ultima Flo AP™ scintillation cocktail (Canberra-Packard, Frankfurt, FRG)
Radiochemicals:
Methyltrienolone, [ 17a-Methyl -3 H] (R 1881) 3089.5 GBq/mmol (NEN Du Pont, Dreieich, FRG)
Fine chemicals, reference:
Dextrane coated charcoal (Sigma, Deisenhofen, FRG)
Methyltrienolone (R 1881) (NEN Du Pont, Dreieich, FRG)
4-Androstene-3,17-dione 98 % (Sigma, Steinheim, FRG)
Dihydrotestosterone (DHT) >99 % (Sigma, Steinheim, FRG)

Details on exposure:

Test compound was incubated overnight with 2 nM androgen receptor and 2 nM radiolabeled methyltrienolone. After removal of unbound ligand by adsorption to charcoal and centrifugation, receptor-bound methyltrienolone was determined by liquid scintillation counting. Incubations were performed in triplicate (control: n = 6 / coefficient of variation =3 %). Receptor binding in the presence of excess (5 µM) unlabeled R1881, i.e., unspecific binding was substracted. Ligand bound in the presence of test compound was related to ligand bound in the absence of compound and expressed as percentage of control.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

overnight

Dose / conc.:

1 other: mmol

Remarks:

Basis:
analytical conc.
test article sodium salt

Control animals:

other: dihydrotestosterone (DHT) and androstenedione (ANDRO)

Details on study design:

In order to screen for a potential affinity of test article sodium salt for the androgen receptor (AR), potential AR binding of test article sodium salt was investigated by means of a classical receptor binding assay using a rat recombinant fusion protein containing both the hinge region and ligand binding domain of the AR as receptor source and radiolabeled methyltrienolone as ligand. The physiological androgens dihydrotestosterone and androstenedione served as reference compounds with strong and weak affinity for the AR, respectively.

Statistics:

no data

Key result

Dose descriptor:

other:

Basis for effect level:

other: No binding to the androgen receptor at the maximally employable concentration of 1 mM.

Remarks on result:

not measured/tested

An affinity of test article sodium salt for the androgen receptor (AR) at the maximally employable concentration of one millimolar could not be demonstrated in two independent experiments. Within the experimental error, the quantity of radiolabeled ligand methyltrienolone bound in the presence of the test compound was comparable to that of the control. The reference compounds dihydrotestosterone (DHT) and androstenedione (ANDRO) reproducibly displaced radiolabeled methyltrienolone from the AR with IC50-values of 4.7 nM and 4.6 nM for DHT and 2.4 pM and 2.0 pM for ANDRO, respectively. In the presence of test article sodium salt generally no displacement of ligand was observed. A slight drop in ligand binding at the maximally testable concentration of 1 mM in one experiment representing the edge of the experimental error, was indistinguishable from that observed in the presence of 1 µM in the same experiment, and thus was not confirmed in the other experiment.

Conclusions:

Under the conditions of this assay system, test article sodium salt was found to have no affinity for the androgen receptor (AR).

Executive summary:

This study was performed to investigate the potential affinity of test article for the androgen receptor (AR) by means of a classical receptor binding assay using a rat recombinant fusion protein containing both the hinge region and ligand binding domain of the AR as receptor source and radiolabeled methyltrienolone as ligand.

Whereas reference compounds dihydrotestosterone and androstenedione reproducibly displaced radiolabeled methyltrienolone from the AR, an affinity of test article sodium salt for the AR at the maximally employable concentration of one mmol could not be demonstrated.

Reference 2

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: scientifically valid method was used and study is adequately reported

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of this in vitro investigation was to evaluate a potential affinity of test article sodium salt for the estrogen receptor (ER). The experimental system employed was capable of detecting strongly and weakly binding compounds such as dihydrotestosterone and androstenedione.
In principle, the method described by Boesel and Shain (Boesel RW, Shain SA, A rapid, specific protocol for determination of available androgen receptor sites in unfractioned rat ventral prostate cytosol preparations. Biochem Biophys Res Comm 61: 1004-1011, 1974) for androgen receptor binding was applied, however, human recombinant estrogen receptor of the α-subtype served as receptor source, whereas radiolabeled estradiol was used as receptor ligand.

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Details on test animals or test system and environmental conditions:

Biological material
Estrogen receptor (ER): human recombinant ER of the α-subtype (PanVera, Madison, Wl, USA)
Equipment:
Labofuge A (Heraeus, Osterode, FRG)
Liquid scintillation counter 1900 TR (Canberra-Packard, Frankfurt, FRG)
Liquid scintillation counter 1450 Microbeta™ Trilux (Wallac, Freiburg, FRG)
Chemicals:
Tris(hydroxymethyl)-aminomethane (Tris) p.a. (E. Merck, Darmstadt, FRG)
Sodium chloride (KMF, St. Augustin, FRG)
Dithiothreitol (DTT) (Sigma, Deisenhofen, FRG)
Glycerol anhydrous pure (E. Merck, Darmstadt, FRG)
Albumin, bovine (BSA) > 96 % (Sigma, Deisenhofen, FRG)
Dimethylsulfoxide p.a. (DMSO) (E. Merck, Darmstadt, FRG)
Ethanol p.a. (E. Merck, Darmstadt, FRG)
Ultima Gold™ scintillation cocktail (Canberra-Packard, Frankfurt, FRG)
Ultima Flo AP™ scintillation cocktail (Canberra-Packard, Frankfurt, FRG)
Radiochemicals:
Estradiol, [2,4,6, 7, 16, 17 -3H (N)], 4366 GBq / mmol (Perkin Elmer, Dreieich, FRG)
Fine chemicals, reference:
Dextrane coated charcoal (Sigma, Deisenhofen, FRG)
Genistein (GEN), 98 % (Sigma, Steinheim, FRG)
(i-Estradiol (E2), 98 % (Sigma, Steinheim, FRG)

Details on exposure:

Test compound as indicated was incubated overnight with nominally 2 nM estrogen receptor and 1.2 nM radiolabeled estradiol. After removal of unbound ligand by adsorption to charcoal and centrifugation, receptor-bound estradiol was determined by liquid scintillation counting. Incubations were performed in triplicate (control: n = 9 / coefficient of variation = 2 %). Ligand bound in the presence of test compound was related to ligand bound in the absence of compound and expressed as percentage of the control.
The value observed in the presence of 0.03 nM estradiol is considered as an outlier.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

overnight

Dose / conc.:

1 other: mmol

Remarks:

Basis:
analytical conc.

Control animals:

other: physiological estrogen estradiol and the phytoestrogen genistein

Details on study design:

In order to screen for a potential affinity of test article sodium salt for the estrogen receptor (ER), potential ER binding of test article sodium salt was investigated by means of a classical receptor binding assay using human recombinant ER of the α-subtype as receptor source and radiolabeled estradiol as ligand. The physiological estrogen estradiol and the phytoestrogen genistein served as reference compounds with strong and weak affinity for the ER, respectively.

Statistics:

no data

Key result

Dose descriptor:

other:

Basis for effect level:

other: No binding to the estrogen receptor at the maximally employable concentration of 1 mM.

Remarks on result:

not measured/tested

The aim of this in vitro investigation was to evaluate a potential affinity of test article sodium salt for the estrogen receptor (ER). The experimental system employed was capable of detecting strongly and weakly binding compounds such as estradiol and genistein. The binding affinity of genistein relative to that of estradiol (ratio of IC50-value estradiol to IC50-value genistein) of 1 % was similar to that recently reported by Kuiper et al. (1997) using a comparable assay system. Within the experimental error, in the presence of test article sodium salt no displacement of ligand was observed.

Conclusions:

Under the conditions of this assay system, the test article sodium salt was found to have no affinity for the estrogen receptor (ER).

Executive summary:

This study was performed to investigate the potential interaction of test article sodium salt with the estrogen receptor (ER) by means of a classical receptor binding assay using human recombinant ER of the α-subtype as receptor source and radiolabeled estradiol as ligand.

Whereas reference compounds estradiol and genistein reproducibly displaced radiolabeled estradiol from the ER, an affinity oftest article sodium salt for the ER at the maximally employable concentration of 1 mmol could not be demonstrated.

Additional information

As discussed above, no embryotoxic or teratogenic effects were noted when pregnant female rats were administered 1000 mg/kg bw/day orally during 10 consecutive days in pregnancy. Also no androgen or estrogen binding effects were seen in such studies nor have any uterotrophic effects been noted in an uterotrophic screening assay. Thus, the substance is considered to not exert any developmental effects. Specific investigation on fertility are not available.

Justification for classification or non-classification

In the absence of teratogenic and embryotoxic effects of the test substance currently there is no indication for classification for reproductive effects according to CLP (Regulation EC No 1272/2008).