Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-10-17 till 2006-05-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
This study was performed in compliance with: Swiss Ordinance relating to Good Laboratory Practice, adopted May 18'" 2005 [RS 813.112.1] This Ordinance was based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MeNigu (1-Methyl-3-nitroguanidin)
- Physical state: solid, white cristals
- Molecular weight: 118.1 g/mol
- Analytical purity: 99.7%
- Lot/batch No.: 9077154
- Expiration date of the lot/batch: June 1, 2007
- Stability under test conditions: test item in feed known to be stable for at least 3 weeks when stored at room temperature
- Storage condition of test material: at room temperature, protected from light and moisture

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Services, Füllinsdorf / Switzerland
- Age at study initiation: 10 weeks (age at pairing)
- Weight at study initiation: 186 - 225 grams (day 0 post coitum)
- Fasting period before study: not reported
- Housing: Animals were housed individually in Makrolon cages with wire mesh tops and standardized granulated softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Kliba-Nafag 3433 ratlmouse maintenance diet was available ad libitum
- Water (e.g. ad libitum): Community tap water from Fullinsdorf in bottles was available ad libitum.
- Acclimation period: Seven days (minimum) prior to pairing under test conditions with an evaluation of the health status.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light / 12 hours dark


IN-LIFE DATES: From: 2005-10-25 To: 2005-11-15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): Daily before administration
- Mixing appropriate amounts with (Type of food): The test item was weighed into a glass beaker on a tared precision balance and the vehicle was
added (wlv). Using an appropriate homogenizer a homogenous mixture was prepared. Separate formulations were prepared for each concentration.
During the daily administration period homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.
- Storage temperature of food: At room temperature (17-23°C)


VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification stated in the study report
- Concentration in vehicle: 0.5% in water
- Amount of vehicle (if gavage): A standard dose volume of 10 mg/kg body weight with a daily adjustment to the actual body weight was used.
- Lot/batch no. (if required): 111953524604357
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for determination of concentration, homogeneity and stability (4 hours) of the dose formulations were taken during the first week of the
administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of the
administration period.
On each occasion three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat
bottomed flasks. Stability samples were taken from the middle only. The samples were frozen (-25°C to -15°C) pending analysis. Analysis was performed using a method provided by the Sponsor (HPLC). The results of the Analytical Phase are presented in the attachment of the study report.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females were housed with sexually mature males in special automatic mating cages, i.e. with synchronized timing to
initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the
different females. The females were removed and housed individually, if a) the daily vaginal smear was sperm positive or b) a copulation plug was
observed. The day of mating was designated day 0 post coitum.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. not applicable
- Further matings after two unsuccessful attempts: [no / yes (explain)] not applicable
- Verification of same strain and source of both sexes: [yes / no (explain)] Male rats of the same source and strain were used for mating only. These male rats were in the possession of RCC.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
15 days
Frequency of treatment:
daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Each group consisted of 22 mated female rats.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a dose range-finding study, where treatment with MeNigu at 100, 300 and 1000 mg/kg body weight/day was well tolerated without any signs of maternal or embryo-fetal toxicity.
- Rationale for animal assignment (if not random): Mated rats were assigned to the different groups using a computer-generated random algorithm. In addition, the body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups on day 0 post coitum.
- Other:

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations checked in table p78 were included.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded daily from day 0 until day 21 post coitum


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: not applicable


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


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: gross macroscopic examination of all internal organs, with emphasis on the uterus, uterine contents, position of fetuses
in the uterus and number of corpora lutea


OTHER:
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: No data
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other:
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
The following statistical methods were used to analyse body weights, food consumption, reproduction and skeletal examination data:
1. Means and standard deviations of various data were calculated and included in the report.
2. If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for
intergroup comparisons (i.e. single treatment groups against the control group).
3. The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
4. Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Indices:
none reported
Historical control data:
Historical control data are attached to the 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:
FOOD CONSUMPTION
In group 4 (high dose group), mean food consumption was statistically significantly decreased throughout the treatment period compared with the control group (-10.7%). In group 3, mean food consumption was only slightly reduced (-3.6%). Mean food consumption in group 2 was not affected by the treatment with the test item.

BODY WEIGHTS
Mean body weights and body weight gains were slightly decreased in group 4 (+39.2% compared to +43.5% in the control group). The mean corrected body weight gain (corrected for gravid uterus weight) was also decreased (+4.6% compared to +7.4% in the control group). In groups 2 and 3, mean body weights were minimal lower throughout the treatment period than in the control group probably due to the smaller number of fetuses per litter (12.0 fetuses each, compared to 13.2 fetuses in the control group). This opinion is supported by the corrected mean body weight gain values, which were similar to that of the vehicle control (+7.6% and +7.9%, respectively).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
>= 100 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
BODY WEIGHT
In group 4, on an individual basis, the mean fetal weights were marginally decreased (-2.1 % for male and female weights combined). As there were also lower number of fetuses per litter compared to the control, the reduced mean fetal weights was considered to be test item related.

SKELETAL EXAMINATION OF FETUSES - STAGE OF DEVELOPMENT
In group 4, when calculated on a fetus basis, skeletal examinations for the stage of development showed statistically significantly increased
incidences of non-ossified cervical vertebral bodies 2, 3 and 4. This slightly lower stage of development was considered to be a consequence
of the maternal toxicity as indicated by the reduced fetal body weights.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Tab. 3: Summary of significant maternal toxic effects

 Observed effect  Control   Test item administration300 mg/kg bw per day  Test item administration1000 mg/kg bw per day  
 Food consumption normalized level -3.6%  -10.7% 
 Body weight gain +43.5%  * +39.2% 
 Corrected body weight gain(corrected for gravid uterus weight)  +7.4% -  +4.6%
  Corrected body weight gain(corrected for gravid uterus weight)  +7.9%  +7.6%  -

* Mean body weights of dose groups 100 and 300 mg/kg bw per day were minimal lower throughout the treatment period than in the control group probably due to the smaller number of fetuses per litter. This opinion is supported by the corrected mean body weight gain values, which were similar to that of the vehicle control.

Tab. 4: Sumary of significant embryotoxic effects

 Observed effect   Control  Test item administration 1000 mg/kg bw per day   
  Body weight gain  normalized level  -2.4% * 
 Skeletal examination  normalized level  statistically significantly increased incidences of non-ossified cervical vertebral bodies

* Additionally smaller no. of embryos per litter, therefore considered test item related effect

Applicant's summary and conclusion

Conclusions:
Based on these results the NOEL for maternal organisms was considered to be 100 mg/kg body weight/day. Based on these results the NOEL for fetal organisms was considered to be 300 mg/kg body weight/day. Under the conditions described for this study MeNigu revealed no teratogenic
potential up to and including the high dose level of 1000 mg/kg body weight/day. No GHS/CLP classification of CA 2342 A under reproductive
toxicity necessary.
Executive summary:

The purpose of this study was to assess the effects of MeNigu on pregnant female rats and embryo-fetal development when administered orally, by gavage, once daily to mated female rats from day 6 through to day 20 post coitum, inclusive (OECD Guideline 414). Each group consisted of 22 mated female rats.

In order to detect effects on the pregnant female and on embryonic and fetal development, MeNigu was administered orally by gavage once daily from day 6 through to day 20 post coitum at dose levels of 0, 100, 300 and 1000 mg/kg body weight/day. A standard dose volume of 10 ml/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (carboxymethylcellulose (0.5% in water). All females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section.

At 1000 mg/kg body weight/day, mean food consumption and body weight gain were statistically significantly decreased throughout the treatment period. At 300 mg/kg body weight/day, mean food consumption was slightly reduced. At 1000 mg/kg body weight/day, the fetal body weights were slightly reduced. Furthermore, ossification of cervical vertebral bodies was slightly less advanced that in the vehicle control.

Based on these results the NOEL (no observed effect level) for maternal organisms was considered to be 100 mg/kg body weight/day. Based on these results the NOEL for fetal organisms was considered to be 300 mg/kg body weight/day. Under the conditions described for this study MeNigu revealed no teratogenic potential up to and including the high dose level of 1000 mg/kg body weight/day.

Comments on GHS/CLP-classification on reproductive toxicity:

The maternal toxicity NOEL of 100 mg/kg bw/d goes back to the slightly decreased food consumption at dose level 300 mg/kg bw/d, an effect which is considered as a minor health effect only. More clearly decreased food consumption, decreased body weight (gain) and slightly less advanced ossification of cervical vertebral bodies of the embryos were observed at the high dose level (i.e.1000 mg/kg bw/d) only. Furthermore there was the total absence of any teratogenic effects at all dose levels. Therefore it is concluded that EU or GHS classification of CA 2342 A as to its reprotoxic properties is not required.