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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-04-22 to 2014-10-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 22 January, 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Guanidinhydrochloride

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: females: 11-12 weeks old (arrival), males: 19-20 weeks old (start of pairing)
- Weight at study initiation: females: 201 - 238 g; males: 330 - 449 g (start of pairing)
- Housing: The animals were kept individually in IVC 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: at least 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: 2014-02-13 to 2014-10-14 females From: 2014-04-15 to 2014-10-14

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: 10 mg/L; medium dose 30 mg/mL; high dose: 70 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:
The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at various intervals.
Samples for analysis of the dose formulations of the test item in the vehicle (nominal concentration) were taken in the first week of the study from the control, LD, MD and HD group (450 mg/kg as well as 350 mg/kg). Samples for analysis of the dose formulations of the test item were also taken in the last week of the study from the control, LD, MD and HD group (350 mg/kg).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day
- M/F ratio per cage: 1:2
The subsequent morning and each morning thereafter, the vaginal smear of each female was checked until positive evidence of mating was confirmed. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’.


Duration of treatment / exposure:
between gestation day 5 until gestation day 19
Frequency of treatment:
7 days per week
Duration of test:
On gestation day 20, sperm positive females were subjected to a caesarean section
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/d
Basis:
actual ingested
Remarks:
Doses / Concentrations:
350 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
control: 21 females
low dose (LD): 23 females
medium dose (MD): 23 females
high dose (HD): 24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
According to the results of the dose range finding study and in consultation with the sponsor the following doses were selected for the 3 dose groups:
LD = low dose: 50 mg/kg bw/d
MD = medium dose: 150 mg/kg bw/d
HD = high dose: 350 mg/kg bw/d
C= control group: vehicle
The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.

Females were paired for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day.

8 females from the HD group were treated with 450 mg/kg body weight/day from the start of the study for several treatment days (until 09 May 2014):
- animal no. 76 from GD 5 to GD 14 (euthanized on GD 14)
- animal no. 77 from GD 5 to GD 17 (found dead on GD 18)
- animal no. 78 from GD 5 to GD 14
- animal no. 79 from GD 5 to GD 10
- animal no. 80 from GD 5 to GD 9
- animal no. 81 from GD 5 to GD 9
- animal no. 82 from GD 5 to GD 8
- animal no. 83 from GD 5 to GD 7
After marked reduction of the body weight and food intake in those presumed pregnant animals of the HD group, the dose of the HD group was reduced to 350 mg/kg body weight/day. Females no. 84-99 of the HD group were treated with a dose of 350 mg/kg body weight/day for the whole treatment period.

- Rationale for animal assignment (if not random):

Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other. Each animal was assigned a unique identification number.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily all animals were observed
- Cage side observations: for morbidity and mortality except on weekends and public holidays when
observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, but no feeding study
-Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No (gavage study)
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: At the time of termination or death during the study, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- 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: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and interpretation. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Historical control data:
Laboratory historical control data for prenatal development toxicity studies - wistar rat (year 2010 - 2014) reported within study report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
One female (no. 76) was euthanized in a moribund condition on gestation day 14 and another female of the HD group (no. 77) was found dead on gestation day 18. The animal of the HD group that was euthanized for welfare reasons on gestation day 14, showed marked loss of body weight, piloerection, moving the bedding and red nasal discharge. The animal of the HD group which was found dead on gestation day 18 lost body weight and was observed with slight to severe piloerection and a wasp waist. Females no. 76 and 77 were administered 450 mg/kg bw/d throughout their test participation.

The clinical sign of slight to severe piloerection was noted transiently in several females of the HD group treated with 450 to 350 mg/kg bw/d whereas only one female of the control group showed piloerection on one single day. Regarding females of the HD group which received 350 mg/kg bw/d throughout the whole treatment period, one female showed slight piloerection on one single day and another female was noted on two single days with slight or moderate piloerection. Thus, these findings in females treated with 450 to 350 mg/kg bw/d can be considered of minor toxicological relevance and in the remaining females as incidental. Slight to severe salivation was noted transiently with a dose-dependent pattern in most females of the HD group (at 450 to 350 mg/kg bw/d as well as at solely 350 mg/kg bw/d) and few females of the MD group. Moving the bedding was observed transiently in all females of the HD group, 10 females of the MD group and one female of the LD group. As the symptoms of salivation and moving the bedding were noted mainly immediately after dose administration, these signs were considered to be indicative of discomfort due to a local reaction to the test item. None of the females showed signs of abortion prior to the scheduled sacrifice.

The mean body weight increased with the progress of the study in the control, the LD and the MD group. However, mean body weight of the HD group was slightly lower compared to the control group from gestation day 8 onwards but without achieving statistical significance. Body weight gain was also slightly lower in the HD group compared to the control group on various intervals within the study period and achieved statistical significance on day 5-8 (p<0.001). The same effect was seen with statistical significance (p<0.01) when considering only those females of the HD group which received a dose of 350 mg/kg bw/d for the whole treatment period. This effect on the body weight and body weight gain of the whole HD group as well as of the HD subgroup receiving a dose of solely 350 mg/kg bw/d was considered to be test item-related.

In correlation to the body weight and body weight gain, food consumption in the HD group was noted to be slightly lower compared to the control group after the beginning of the treatment. Statistical significance was noted over the study period from day 0 to day 20 (p<0.001) as well as on several treatment intervals. Food consumption was comparably reduced when considering only those females of the HD group with a continuous dosage of 350 mg/kg bw/d. Likewise, statistical significance was noted over the study period from day 0 to day 20 (p<0.01) as well as on several treatment intervals. This effect on the food consumption of the whole HD group as well as of the HD subgroup receiving a dose of solely 350 mg/kg bw/d was considered to be test item-related.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no test item-related effects of toxicological relevance noted on mean foetus weight, total number of foetuses, number of male and female foetuses, mean total litter weight and male and female litter weight.

There were no external abnormalities considered to be of toxicological relevance in any of the dose groups.

A range of visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls and litter incidences were statistically insignificant except for liver haematoma. Liver haematoma was noted with a statistically significantly higher litter incidence in the control group compared to the LD group without dose-dependency (C: 22%, LD: 0%, MD: 14%, HD: 10%). This was considered as incidental and not related to the treatment with the test item. As the remaining observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

Craniofacial examination by a razor blade serial sectioning technique revealed few findings at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to the controls. Statistical analysis of the data revealed no significant effect in any of these findings as compared to the control group. Therefore, these findings are not to be considered to be treatment related and spontaneous in nature.

Skeletal examination of the Alizarin red stained foetuses revealed a range of abnormalities which were of a type or which occurred at an incidence comparably lower in treated groups when compared to the control group. Statistically significant decrease in litter incidence of incomplete ossification of frontal bone (B) in HD and of 4th sternebra in HD, 14th full rib (B) in LD and supernumerary rib cartilage (B) in LD was observed when compared to the control group. Lack of dose dependency and consistency in these findings indicated no relevance with treatment. There was no indication of a compound-related trend in the type and incidences of other abnormalities and they were therefore considered to be spontaneous in nature.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Prenatal parameters like group mean gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, number of live and dead foetuses, number of early and late resorptions, number of male and female foetuses, sex ratio and pre- and post-implantation loss remained unaffected in the dose groups when compared to the control group. Pregnancy rates were comparable between the groups and within the normal range of variation for animals of this strain and age.

Applicant's summary and conclusion

Conclusions:
Under the condition of the study, 150 mg/kg body weight/day was considered as no observed adverse effect level (NOAEL) for maternal toxicity and 350 mg/kg body weight/day for developmental toxicity of Guanidine hydrochloride.
Executive summary:

In a developmental toxicity study according to OECD guideline 414 (adopted 22 January, 2001), Guanidine hydrochloride was administered to female Wistar rats during gestation days 5 to 19.

The following doses were evaluated:

Control (C): 0 mg/kg body weight/day (21 females)

Low Dose: 50 mg/kg body weight/day (23 females)

Medium dose (MD) 150 mg/kg body weight/day (23 females)

High dose (HD): 350 mg/kg body weight/day (24 females)

Females were paired (ratio 1:2) for cohabitation in batches in order to regularise the number of animals for terminal sacrifice on a particular day.

At a dose level of 450 mg/kg bw/d mortality was observed in two animals and clinical symptoms occurred in more animals than in the control group resulting in a reduction of the highest dosage to 350 mg/kg bw/d. Animals treated with 450 to 350 mg/kg bw/d as well as animals receiving 350 mg/kg bw/d throughout the whole treatment period were noted with a statistically significantly reduced body weight gain and food consumption.

No effect on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level.

Maternal toxicity: NOAEL: 150 mg/kg bw/day

Based on mortality at 450 mg/kg bw/day; statistically significantly reduced body weight gain and food consumption (450 and 350 mg/kg bw/day)

Developmental toxicity: NOAEL: 350 mg/kg bw/day

No effects on prenatal data, litter data, foetal external, visceral, skeletal and craniofacial parameters were observed up to the highest dose level