Guanidinium chloride

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-03-27 to 2015-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents), adopted 21 September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: females: 7-8 weeks old, males: 7-8 weeks old
- Weight at study initiation: females: 137 -170 g; males: 147 -193 g
- Housing: The animals were housed in groups (5 animals/sex/cage) in type IV cages
- Diet (e.g. ad libitum): Altromin 1324 maintenance diet for rats and mice (lot no. 1526) ad libitum
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

IN-LIFE DATES: males From: 2014-03-25 to 2014-08-05 females From: 2014-03-25 to 2014-08-04

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
-aqua ad injectionem (AlleMan Pharma)
- Concentration in vehicle: low dose: 5 mg/L; medium dose 20 mg/mL; high dose: 60 mg/mL
- Amount of vehicle (if gavage): dose volume for all groups was 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For determination of the concentration of test item in dosing formulations, samples of at least 5 mL were retained from all groups in weeks 1, 5, 9 and 13
during the treatment period and stored between -15 and -35 °C. In total 16 samples.
Stability of the dosing formulations was tested once at the beginning of the treatment period.
From the low, medium and high dose group, samples of dosing formulations were frozen after 0 hours and after 10 days (at room temperature) after the preparation and stored at -15 to -35 °C until analysis. In total 6 samples.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 days per week

Details on study schedule:

Fertility parameters from a subchronic repeated dose toxicity study

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Medium dose

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

control: 15 animals per sex
high dose: 15 animals per sex
low dose: 10 animals per sex
mid dose: 10 animals per sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: according to the results of a previous dose range finding study
- Rationale for animal assignment (if not random): Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively, while ensuring to keep each animal with its initial cage partners.
- Rationale for selecting satellite groups: In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects, the animals in the recovery groups were observed for a period of 28 days (females) or 29 days (males) following the last administration.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

Refer to entry under IUCLID section 7.5.1

Oestrous cyclicity (parental animals):

Daily over a period of 8 days, the estrous cycle of all female animals was examined 4, 8 and 12 weeks after the first administration.
In the recovery animals the estrous cycle was examined during the last week of the recovery period.

Sperm parameters (parental animals):

At necropsy (one day after the last administration) and at the end of the recovery period, left epididymis, left testis and left vas deferens were separated and used for evaluation of sperm parameters.
Epididymal sperm motility and testicular sperm count were evaluated in all male animals using Hamilton Thorne Sperm Analyser
(TOX IVOS Version 13.0).
Therefore sperm from left vas deferens was transferred to 0.1% bovine serum albumin solution. For staining two drops of 1% aqueous Eosin-Y solution were mixed with six drops of the sperm-suspension. The stained sperm suspension was used to prepare smears on slides. After complete drying the slides were dipped into 0.1% acetic acid for approximately 30 seconds to intensify the colouring.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed occasionally, mainly as irregularly long cycles, in all treatment groups including control and irrespective of the duration of the treatment. This was considered incidental and not related to the treatment with the test item.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. The statistical analysis showed no statistically significant changes between the control group and any of the dose groups neither in the percentage of motile, static or rapidly moving epididymal sperms nor in testicular number of sperms/g testis.
Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item.

For other parameters refer to IUCLID section 7.5.1 Robust Study Summary for repeated dose toxicity

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

In a subchronic toxicity study according to OEDC Guideline 408 (adopted 21 September 1998), Guanidine hydrochloride was administered to 10 Wistar rats/sex/dose in water, by gavage at dose levels of 0, 50, 100 and 300 mg/kg bw/day.

In order to allow a detection of possible delayed occurrence or persistence of or recovery from toxic effects a satellite group of 5 rats/sex was exposed at dose levels of 0 and 300 mg/kg bw/day (control and HD).

To evaluate possible toxic effects on fertility, the estrous cycle was examined at defined time points of the treatment and recovery period and epididymal sperm motility, testicular sperm count and sperm morphology from vas deferens were evaluated at the end of the treatment and recovery period. Moreover, a detailed histopathological evaluation of the reproductive organs was performed.

Guanidinhydrochloride had no effect on epididymal sperm motility or testicular sperm count analyzed at the end of the treatment or recovery period of this study. Sperm staging and evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item. Guanidinhydrochloride had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration and in the last week of the recovery period.