Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-04-20 till 2007-02-01
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:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
This study was conducted in compliance with the Good Laboratory Practice Regulations based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997
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
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Services, Füllinsdorf / Switzerland
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) Males: 203 - 244 grams, Females: 132 - 173 grams
- Fasting period before study: not reported
- Housing: Individually in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): Pelleted standard Kliba-Nafag 3433 rat/mouse 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 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 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light / 12 hours dark


IN-LIFE DATES: From: 2006-04-20 To: 2006-08-28

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): Frequency of preparation at least every 2 weeks
- Mixing appropriate amounts with (Type of food): The test item was mixed to granulated diet pelleted in a pelleting machine. Water was added to
each feed preparation at a volume/weight ratio of approximately 1:10 to ensure pelleting, after which the pellets were dried with warm air for
approximately 48 hours before storage.
- Storage temperature of food: The dietary admixtures were stored in closed stainless steel containers at room temperature (17 - 23°C).


VEHICLE
not aplicable
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: usually 14 days, maximum of 21 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as day 0 post coitum
- After 21 days of unsuccessful pairing, replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): After mating or at the end of the pairing period, males and females were housed
individually, males until necropsy and females for the birth and rearing of young.
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analyses of test item, its content and homogeneity in the feed were drawn at the start of the prepairing period and at the end of gestation/start of lactation period (i.e. two occasions). The analyses were performed using an external standard method (HPLC). Stability (3 weeks) of the test
item in the feed was determined at the start of the prepairing period (i.e. one occasion). For assessment of content and homogeneity, a 100 g sample was collected from each the top, middle and bottom of every dietary admixture of the respective diet preparation. Samples for assessment of contentand homogeneity were collected after 48 hours of drying the prepared pellets and retained frozen between ca. -24 and -16°C prior to analysis. For
assessment of stability, a 100 g sample was drawn from the middle of the dietary admixture after 48 hours of drying the prepared pellets, stored at
room temperature for 3 weeks and consequently frozen as described above.
Duration of treatment / exposure:
Males: The test item was administered during a 70-day prepairing period, during the pairing period and the after pairing period until necropsy.
Females: The test item was administered during a 70-day prepairing period, and also during the pairing, gestation and lactation periods until day 21 post partum, one day before necropsy.
Frequency of treatment:
daily administration of test item through feeds
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters. not applicable
- Selection of parents from F1 generation when pups were [...] days of age. not applicable
- Age at mating of the mated animals in the study: approx. 16 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
640 mg test item per kg feed
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
3200 mg test item per kg feed
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
16000 mg test item per kg feed
Basis:
nominal in diet
No. of animals per sex per dose:
24 P-Generation males and 24 P-Generation females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on a dose range-finding study, where the limit dose of 1000 ppm/body weight was not attained
with 12000 ppm in the males, but only 796 ppm in the prepairing period and 802 ppm in the after pairing period. Furthermore, because of the
unclear effects on body weight development of males already at 1200 ppm, the concentration of the lower dose groups was reduced.
- Rationale for animal assignment (if not random): Prior to start of treatment, P animals were assigned to the different groups using a computer-
generated random algorithm. In addition, body weights were taken into consideration in order to ensure similar mean body weights in all groups.
- Other:
Positive control:
not reported

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations checked in table p100ff were included. All animals were checked at least twice daily for morbidity or mortality.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice daily for signs of reaction to treatment and/or symptoms of ill health.


BODY WEIGHT: Yes
- Time schedule for examinations: P generation animals were weighed at weekly intervals, with the exception of the pairing period. After mating,
females were weighed on days 0, 7, 14 and 21 post coitum. Dams which littered were weighed on days 1,4,7, 14 and 21 post partum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption was measured weekly together with the recording of body weights, except during the pairing period when no food consumption was measured. During the lactation period, food consumption was recorded only until day 14 post partum. Relative food consumption ratios
expressed as ppm was calculated.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


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


OTHER:
Oestrous cyclicity (parental animals):
Estrous cycles of 4-5 days duration were noted during the study
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- Testis weight, prostate weight, epididymis weight, cauda epididymis weight
- The respective ORGAN/BODY WEIGHT RATIOS (%) were calcultaed
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter, equally divided as to sex, where possible; excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals. P generation males were sacrificed after weaning.
- Maternal animals: All surviving animals. P generation females were sacrificed on day 22 p.p.


GROSS NECROPSY
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
The following organ weights were recorded from all P animals on day 22 post partum. Paired organs were weighed individually: Seminal vesicles, with coagulating glands and their fluids (as one unit), Epididymides (total weight and cauda separately), Testes, Prostate, Ovaries, Uterus (including cervix, excluding oviducts)
Descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected from all parent animals at necropsy and, unless otherwise indicated, fixed in neutral phosphate buffered 4% formaldehyde solution:
gross lesions, adrenals, ovaries, pituitary, prostate, seminal vesicles with coagulating gland, testes with epididymides (in Bouin's fixative), uterus and cervix, vagina, target organs

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table p117ff, p278ff, and p428ff were prepared for microscopic examination and weighed, respectively. Slides of all organs and tissues collected at necropsy (see list above) from all animals of the control and high dose group (group 1 and 4, respectively) and all gross lesions from all animals of all groups were processed, embedded in paraffin, cut at a nominal thickness of 2-4 micrometers, stained with hematoxylin & eosin (H&E) and examined by light microscope by the study pathologist.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at day 4 post partum (day of standardization of no. of pups), or on day 21 p.p., respectively
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: organs, disclorations, injuries, irregular hair growth, sex ratio, weight, etc.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY / ORGAN WEIGTHS
No histopathology / organ weights reported for F1 offspring
Statistics:
The following statistical methods were used to analyse body weights, food consumption, reproduction and breeding data:
1. Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
2. If the variables were assumed to follow a normal distribution, the Dunnett manyone 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.
Reproductive indices:
Mating performance, fertility, breeding data per group - all dams giving birth, breeding data per group - all dams weaning young, estrous cycles
Offspring viability indices:
Dates of postnatal losses of F1 pups during lactation

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths due to the treatment with the test item and no signs of reaction to treatment were noted.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)

1. Body weight
- Males prepairing period: In group 4, mean body weight and mean body weight gain were statistically significantly reduced throughout
the prepairing period (+79.6% compared to +93.0% in the control group). This reduction was considered to be test item-related. In groups 2 and 3,
mean body weight and mean body weight gain were similar to the control group.
- Females prepairing period: In group 4, mean body weight gain was statistically significantly reduced throughout the prepairing period
(+58.6% compared to +66% in the control group). Mean body weight was minimal and only towards the end of the prepairing period reduced. These
reductions were considered to be test item-related. In groups 2 and 3, mean body weight and mean body weight gain were similar to the control
group.

2. Food consumption:
- Males prepairing period: in group 4, mean food consumption was slightly, statistically significantly reduced until day 36 of the prepairing period
(-7.1% compared to the control during days 29-36). This reduction was considered to be test item-related. In groups 2 and 3, mean food
consumption was similar to the control group.
Females:
- Females prepairing period: In group 4, mean food consumption was slightly, statistically significantly reduced during the first 8 days of the
prepairing period (-8.9% compared to the control group). This reduction was considered to be test item-related. In groups 2 and 3, mean food
consumption was similar to the control group.

3. Relative food consumption:
- Males prepairing period: In group 4, mean relative food consumption was statistically significantly reduced during the first 8 days of the prepairing
period. After day 36 of the prapairing period onwards, mean relative food consumption was statistically significantly increased, due to the lower
mean body weights. Both findings were considered to be test item-related. In group 2, mean relative food consumption was similar to the control
group. In group 3, mean relative food consumption was statistically significantly increased after day 22 of the prepairing period onwards, due to
slightly higher absolute mean food consumption of the males. This increase was considered to be incidental.
- Females prepairing period: In group 4, mean relative food consumption was statistically significantly reduced during the first 8 days of the
prepairing period. After day 43 of the prapairing period onwards, mean relative food consumption was statistically significantly increased, due to the lower mean body weights. Both findings were considered to be test item-related. In groups 2 and 3, mean relative food consumption was similar to
the control group.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Overall mean test item intakes during the whole investigation period corresponded to the order of ascending dose levels.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Vaginal estrous cycles at all dosages were unaffected by treatment with the test item. The mean duration of gestation was unaffected by treatment
with the test item. Mean duration of gestation was 21.5, 21.7, 21.5 and 21.8 days, in order of ascending dose level.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
not reported

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating index (no. of animals with seminal plug or sperm / no. mated x 100): 100%: All females were mated during the 14-day pairing period.
Fertility index (no. of animals with implants / no. of matings x 100): The calculated fertility indices were 91.3, 95.8, 87.5 and 87.5, in order of
ascending dose level, due to 2, I , 3 and 3 non pregnant females.
Gestation index (no. of delivering females / no. of females with implant): The gestation index was 100% in all dose groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No test item-related effects on organ weights were noted in any dose group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No abnormal findings that were considered to be test item-related were noted during macroscopic examination.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no findings, which distinguished test item-treated animals from controls. In particular, no treatment-related histopathological findings
were observed in the reproductive organs of either sex from the parental generation.

OTHER FINDINGS (PARENTAL ANIMALS)

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
>= 3 200 mg/kg diet
Sex:
male/female
Basis for effect level:
other: At 16000 ppm, mean food consumption was reduced during the first half of the prepairing period and mean body weight and mean body weight gain were reduced throughout the treatment period.
Dose descriptor:
NOEL
Effect level:
>= 16 000 mg/kg diet
Sex:
male/female
Basis for effect level:
other: Based on the absence of any effects on reproductive parameters the NOEL for reproductive performance was considered to be 16000 ppm.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was 11.8, 11.2,
11.9 and 10.7, in order of ascending dose level. In group 1, one dam had 5 dead pups only at first litter check.

CLINICAL SIGNS (OFFSPRING)
At first litter check, 1, 2 and 3 pups had no milk in the stomach in groups 1, 2 and 4. Most of these pups were found dead. No findings that were
considered to be test item-related were noted at first litter check or during lactation. Additionally, the following incidental findings were found in one pup each in groups 1-4, respectively: a bluish discoloured nose, a blue discoloured tip of tail, a dark discoloured body and one pup was partially
cannibalized.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item.
Mean pup weight development during lactation was statistically significantly decreased from day 14 onwards in group 4 compared to the control
group (27.7 g versus 30.2 g on day 14 of the lactation period, combined data for male and female pups). Mean pup weights on day 21 post partum
were 49.1. 50.7, 48.2 and 44.9 g in order of ascending dose level. This reduction in mean body weights of pups in group 4 was considered to be
test item-related.

SEXUAL MATURATION (OFFSPRING)
Sex ratios at first litter check and on day 21 post partum were unaffected by treatment with the test item.

ORGAN WEIGHTS (OFFSPRING)
not examined

GROSS PATHOLOGY (OFFSPRING)
In group 1, for one pup the left kidney was missing. No other findings were noted during necropsy in any group.

HISTOPATHOLOGY (OFFSPRING)
not examined

OTHER FINDINGS (OFFSPRING)
The mean number of implantations per group and post-implantation loss were unaffected by treatment with the test item. The mean numbers of
implantations per litter were 13.3, 12.6, 13.1 and 12.1, in order of ascending dose level. Post-implantation losses as a percentage of total
implantations were 11 . I , 10.7, 9.4 and 11.4%, in order of ascending dose level.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 3 200 mg/kg diet
Sex:
male/female
Basis for effect level:
other: Mean pup weight development during lactation was decreased at 16000 ppm from day 14 post partum onwards.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Tab. 4: Summary results on body weights

 Generation / investigation period  Parameter Sex   16000 ppm  Control 
 P-animals / prepairing period  Weight gain  M  +79.9%  +93.0%
  P-animals / prepairing period   Weight gain  F  +58.6%  +66.0%
  P-animals / prepairing period  Food consumption  M -7.1%   normalized zero level
  P-animals / prepairing period    Food consumption  F -8.9%   normalized zero level
F1 / day 14 post partum   Weight  M/F  27.2 g  30.2 g
  F1 / day 21 post partum    Weight  M/F  44.9 g  49.1 g

For both males and females during prepairing period, in group 4 (16000 ppm test item in food), mean relative food consumption was statistically significantly reduced during the first 8 days of the prepairing period. After day 36 / 43 of the prapairing period onwards (for males / females. respectively), mean relative food consumption was statistically significantly increased, due to the lower mean body weights. Both findings were considered to be test item-related.

Applicant's summary and conclusion

Conclusions:
Based on these data, for the test item MeNigu a NOEL for parental animals and F1 pups was established at 3200 ppm.
Based on the absence of any effects on reproductive parameters the NOEL for reproductive performance was considered to be 16000 ppm.
Executive summary:

The purpose of this study (acc. to OECD Guideline 415) was to provide general information concerning the effects of the test item on reproductive function as assessed by gonadal function, estrus cycles, mating behavior, conception, parturition, lactation, weaning, and the growth and development of the off-spring. The study also provides information about the effects of MeNigu on neonatal morbidity, mortality, development and preliminary data on teratogenesis and to serve as a guide for subsequent tests.

MeNigu was administered per feed to males for a 70-day pre-pairing period, during the pairing period and after pairing period. Females received the test item during a 70-day prepairing period and also during the pairing, gestation and lactation periods. The following dietary concentrations were applied: Group 1: 0 ppm (control) Group 2: 640 ppm Group 3: 3200 ppm Group 4: 16000 ppm. Control animals received the same diet but without the test item. The mean test item intakes for the high dose group (16000 ppm test item per kg feed) were found to be higher than 1000 mg/kg body weight/day, which is the so-called limit dose according to OECD Guideline 415.

On day 4 post partum, the number of offspring was reduced to 8 per litter, where possible. P generation males were sacrificed after weaning of the pups. All surviving dams were sacrificed on day 22 post partum and pups on day 21 post partum.

Treatment with the test item was well tolerated in all P generation animals. No test item-related mortalities, no signs of discomfort or clinical signs of reaction to treatment were observed in any group. At 16000 ppm, mean food consumption was reduced during the first half of the prepairing period and mean body weight and mean body weight gain were reduced throughout the treatment period in males and females. Mean pup weight development during lactation was decreased at 16000 ppm from day 14 post partum onwards. No further adverse effects of the test item was observed, particularly concerning reproductive functions and reproductive performance.

Based on these data a NOEL (no observed effect level) for parental animals and F1 pups was established at 3200 ppm. Based on the absence of any effects on reproductive parameters the NOEL for reproductive performance was considered to be 16000 ppm.

Comments on GHS classification:

The NOEL of 3200 mg/kg feed was due to reduced food consumption (P) and reduced body weight / body weight gain (P and F1) at dose group 16000 mg/kg feed. This can be seen as a minor effect, considering that it occured at the high dose level of actual test item intake > 1000 mg/kg body weight/day. Furthermore there was no effect on reproductive performance. In the case of substances of low toxicity as demonstrated in repeated-dose studies, if a dose of at least 1000 mg/kg bw/d produces no evidence of interference with reproductive performance, studies at other dose levels may not be considered necessary. Therefore it is concluded that EU- or GHS-classification of CA 2342 A as to its reprotoxic properties is not required.