N-nitro-N-(3-methyl-3,6-dihydro-2H-1,3,5-oxadiazin-4-yl)amine

Toxicity to reproduction

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:

March 2008 - March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study according to OECD 415

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC 88/302: One-Generation Reproduction Toxicity Test L 133/43, 1988

Deviations:

no

GLP compliance:

yes

Remarks:

This study was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [RS 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-060-1

EC Name:

-

Cas Number:

153719-38-1

Molecular formula:

C4H8N4O3

IUPAC Name:

3-methyl-N-nitro-3,6-dihydro-2H-1,3,5-oxadiazin-4-amine

Details on test material:

- Name of test material (as cited in study report): CA 2343A (Methyl-Nitro-Oxadiazinamine - Intermediate of Thiomethoxam)
- Physical state: solid, fine white crystals
- Analytical purity: 99.3 %
- Batch No.: 427801
- Expiration date of the lot/batch: 31 July 2010
- Stability of test item in vehicle: 7 days in 0.5% CMC at room temperature
- Storage condition of test material: 20 +/- 5 °C

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd., Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Strain: HanRcc: WIST(SPF)
- number of animals: 96 males (24 per group), 96 females (24 per group)
- Age at study initiation: (P) Males: 8 wks; Females: 9 wks
- Status: Helthy; females nulliparous and non-pregnant
- Weight at study initiation: (P) Males: 236-279 g; Females: 163-203 g;
- Fasting period before study: none
- Housing: animals were housed individually in Macrolon cages type 3 with wire mesh tops and standard soft wood bedding ('Lignocel' Schill AG, 4132 Muttenz/Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: ad libitum, cetrified pelleted standard diet Kliba Nafag 3433 rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles
- Acclimation period: at least 7 days before treatment start
- dose group assignment: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Identification: P animals: Cage card and individual animal number (ear tattoo). Pups: On day 1 post partum, pups were individually tattooed with Indian ink. At the onset of hair growth, pups were identified by color spots on the fur.


ENVIRONMENTAL CONDITIONS
- standard laboratory conditions
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation of diet (frequency): The dose formulations were prepared weekly and split into 7 daily aliquots for dosing.
- CA2343A (Methyl-Nitro-Oxadiazinamine-Intermediate of Thiamethoxam) was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
- Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
- Storage of dose formulations: Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.
VEHICLE
- Identification: 0.5% CMC (carboxymethylcellulose), medium viscosity
- Source: Fluka
- Description: Clear fluid
- Batch number: 1367388 34707017
- Expiry date (retest date): 28-Feb-2018
- Storage conditions: Room temperature (20 ± 5 °C)
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 40 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg body weight

Details on mating procedure:

MATING PROCEDURES
During the pairing period, females were housed with sexually mature males (1:1) 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) A copulation plug was observed, and / or
b) The daily vaginal smear was sperm positive.
The day of mating was designated day 0 post coitum.
All dams were allowed to give birth and rear their litters (F1 pups) up to day 21 post partum. Day 0 was designated as the day on which a female had delivered all her pups. The offspring were examined as soon as possible after completion of delivery for litter size, number of live and still births, and any gross anomalies. On day 4 post partum, the number of offspring was reduced to 8 per litter (when possible equally divided as to sex). On the day of weaning (day 21 post partum), the dams were separated from their litters.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs and 7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration.
- The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. D. Flade (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis, according to a HPLC method provided by the Sponsor. It had to be ensured that the dose formulations were re-suspended after thawing and prior to analysis.

Duration of treatment / exposure:

MALES: minimum 11 weeks
FEMALES: approx. 11 weeks

Frequency of treatment:

Daily

Details on study schedule:

see table 1 below (study schedule, schematic diagram)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 (control)
Basis:
nominal conc.
mg/kg/day

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50
Basis:
nominal conc.
mg/kg/day

Doses / concentrations 3

Remarks:

Doses / Concentrations:
150
Basis:
nominal conc.
mg/kg/day

Doses / concentrations 4

Remarks:

Doses / Concentrations:
500
Basis:
nominal conc.
mg/kg/day

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of previous dose range finding studies available in Sponsor’s files.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

Viability / mortality:
- Twice daily

Clinical signs:
- Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
- Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

Food consumption:
- Males: Weekly during pre-pairing and after pairing periods.
- Females: Weekly during pre-pairing, gestation and lactation periods.
- No food consumption was recorded during the pairing period.

Body weight:
- Recorded daily from treatment start to day of necropsy.

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

- Number of missing (cannibalized) or dead pups: daily.
- Sex ratio Days 0/1, 4 and 21 post partum
- Body weight Days 0/1, 4, 7, 14 and 21 post partum

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: sacrificed when they were no longer needed for the assessment of reproductive effects.
- Maternal animals: At or shortly after weaning

PARENTAL NECROPSY OBSERVATIONS
- All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77 ®).
- All P generation animals were exsanguinated.
- All parent animals were examined macroscopically for any structural changes at the scheduled necropsy. Special attention was directed at the organs of the reproductive system.
- Implantation sites were counted for all dams. The uteri were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
- Samples of the following tissues and organs were collected from all P animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution:
Gross lesions
Ovaries
Pituitary
Prostate
Seminal vesicles with coagulating gland
Testes with epididymides (in Bouin’s fixative)
Uterus and cervix
Vagina
- Slides of all organs and tissues collected at terminal sacrifice from the animals in the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.
- Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of infertile males was made.

Postmortem examinations (offspring):

SACRIFICE
- The remainig pups were sacrificed at or shortly after weaning.

PARENTAL NECROPSY OBSERVATIONS
- All animals were sacrificed by an injection of sodium pentobarbital (Eutha 77 ®).
- For standardization of litter sizes, F1 pups were sacrificed on day 4 post partum, examined macroscopically for possible defects and preserved in neutral phosphate buffered 4% formaldehyde solution.
- All the remaining pups were sacrificed and examined macroscopically for defects after weaning.
- Dead young, except those excessively cannibalized, were autopsied and/or preserved in fixative for possible further examination.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• All statistical tests were two-sided.
• Statistical significance between groups was evaluated by Analysis of Variance (ANOVA). In the case where variances were non-homogeneous, appropriate transformations were applied (e.g. log, square root, or double arcsine) to stabilize the variances before ANOVA. The Dunnett many-one t-test was then used to compare each group to the control based on the error mean square in the ANOVA.
• Fisher's exact-test was applied to the macroscopical findings.

Reproductive indices:

- Mating performance and fertility (mean precoital time)
- Mean duration of gestation
- Mean number of implantations per dam
- Mean post-implantation loss
- Mean number of pups
- Post-natal loss
- Breeding loss

Offspring viability indices:

PUP OBSERVATIONS AND MEASUREMENTS (each pup was individually assessed as follows)
- viability/mortality: determined as viable or stillborn on day 0 p.p.; mortality checked daily thereafter
- sex: determined by external examination on day 0 p.p.
- Findings at birth (first litter check) and during lactation to weaning: external examination of F1 pups at first litter check after parturition, clinical signs or observations of F1 pups during lactation, dates of postnatal losses of F1 pups during lactation and individual pup data
- Sex ratios: Breeding data per group
- body weight (day 21 post partum)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

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:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All animals survived until the scheduled necropsy.
All males in the 500 mg/kg/day group were sedated on the first day of dosing and had ruffled fur from day 2 - 15 of the pre-pairing period. From day 11 onwards, all males pushed their head through the bedding after dosing. In the females, sedation was observed on day 1 of the pre-pairing period and ruffled fur for up to 3 days. From day 7 onwards, all females pushed their head through the bedding after dosing. All these findings were considered to be related to the treatment with the test item. However, pushing the head through the bedding was considered to be a sign of discomfort but not a toxic effect. Two females at this dose level had hair loss and one female and 2 males had salivation for a short period of time during the
study. The hair loss and salivation were considered to be isolated and incidental findings.
All males in the 150 mg/kg/day group were sedated on the first day of dosing. Thereafter, up to day 6 of the pre-pairing period, all males had ruffled fur. The females had sedation and ruffled fur on day 1 of the pre-pairing period. Thereafter isolated, incidental findings such as a missing upper tooth, salivation and a wound on the shoulder were noted in the males during the study.
No test item-related findings were observed in the males and females in the 50 mg/kg/day group. One male had a wound on the shoulder. No other clinical signs were noted for any male or female during the study.
In the control group, no clinical signs were noted for any male during the study. One female
had hair loss during the study.


FOOD CONSUMPTION (MALES)
PRE-PARING PERIOD:
Mean food consumption was dose dependently and statistically significantly decreased in the 50, 150 and 500 mg/kg/day groups during the first week of treatment. Thereafter there were no adverse effects on food consumption.
AFTER PARING PERIOD:
During the after pairing period, mean food consumption was considered not to have been affected by treatment with the test item at any dose level.


BODY WEIGHT (MALES)
PRE-PARING PERIOD:
In the 150 and 500 mg/kg/day groups, mean body weight and body weight gain were dosedependently and statistically significantly reduced during the whole of the pre-pairing period. At the end of the pre-pairing period body weights were 6% and 17% below control in the 150
and 500 mg/kg/day groups respectively.
In the 50 mg/kg/day group there was a transient reduction in body weight and body weight gain at the start of the dosing period. The maximum difference in body weight from control was 4%.
PARING PERIOD:
Body weights of males in the 150 and 500 mg/kg/day groups remained statistically significantly lower than control throughout the pairing period. Body weight gains were similar to or slightly higher than those of controls.
There were no statistically significant differences from control in body weight or body weight gain in the 50 mg/kg/day group.
AFTER PARING PERIOD:
During the after pairing period, body weight gain was not considered to have been affected by treatment with the test item. Body weight in the 500 mg/kg/day group remained statistically significantly reduced. In the 50 and 150 mg/kg/day groups, body weight remained reduced but without statistical significance.


FOOD CONSUMPTION (FEMALES)
PRE-PARING PERIOD:
In the 500 mg/kg/day group, mean food consumption was statistically significantly reduced for the first 3 weeks of the pre-pairing period.
In the 150 mg/kg/day group, food consumption was statistically significantly reduced for the first 2 weeks of the pre-pairing period.
In the 50 mg/kg/day group, mean food consumption was similar to that of the control group.
GESTATION PERIOD:
Mean food consumption was not affected by treatment with the test item at any dose level during the gestation period.
LACTATION PERIOD:
In the 500 mg/kg/day group, mean food consumption was statistically significantly reduced during the second and third week of the lactation period
Mean food consumption was slightly lower than control in the 150 mg/kg/day group in weeks 2 and 3, although the differences were not statistically significant.
In the 50 mg/kg/day group, mean food consumption was not affected by treatment with the test item.

BODY WEIGHT (FEMALES)
PRE-PARING PERIOD:
In the 500 mg/kg/day group, mean body weight and body weight gain were statistically significantly reduced throughout the pre-pairing period.
In the 150 mg/kg/day group, mean body weight and body weight gain were reduced throughout the pre-pairing period and were statistically significant for the majority of the period.
In the 50 mg/kg/day group body weight and body weight gain were similar to control.
PARING PERIOD:
Mean body weight gain was considered not to have been affected by treatment with the test item. Mean body weight remained statistically significantly reduced in the 500 mg/kg/day group (days 1-3) and reduced in the 150 mg/kg/day group. However, the majority of the females only remained in the pairing period for a short period of time before pairing took place.
GESTATION PERIOD:
Mean body weight throughout gestation and mean body weight gain from day 15 of gestation were statistically significantly reduced in the 500 mg/kg/day group.
In the 50 and 150 mg/kg/day groups, mean body weight gain was similar to that of the control group. Mean body weight was occasionally statistically significant reduced in the 150 mg/kg/day group.
LACTATION PERIOD:
In the 500 mg/kg/day group, mean body weight gain was statistically significantly increased for the last 3 days of the lactation period but body weight remained statistically significantly reduced throughout the period.
In the 50 and 150 mg/kg/day groups, body weight gain was similar to that of the control group. Mean body weight remained reduced in the 150 mg/kg/day group, whilst in the 50 mg/kg/day group it was similar to that of the control group.


MATING AND FERTILITY
- Mean precoital time was similar in all dose groups. Two dams in the 50 mg/kg/day group and 2 in the 150 mg/kg/day group were not pregnant. This was considered to be incidental.
- Mean duration of gestation was similar in all dose groups.


PREGNANCY AND DELIVERY
- In the 500 mg/kg/day group, the mean number of implantations per dam was statistically significantly reduced (11.1 compared to 13.8 in the control group). This was considered to be a test item-related effect. Mean post-implantation loss was the same as in the control group. In the 50 and 150 mg/kg/day groups, the number of implantations and post-implantation loss was the same as in the control group.
- The mean number of pups at the first litter check was statistically significantly reduced in the 500 mg/kg/day group.
- In the 50 and 150 mg/kg/day groups, the mean number of pups was not affected by treatment with the test item.
- Post-natal loss was statistically significantly increased in the 500 mg/kg/day group. This increase was partly due to dam no. 179, which lost all 12 pups. In the 50 and 150 mg/kg/day groups, post-natal loss was not affected by treatment with the test item.
- Two pups from two separate litters in the 500 mg/kg/day group were lost between days 5 and 21. All pups in the control, 50 and 150 mg/kg/day groups survived from day 5 to 21


ORGAN WEIGHTS (PARENTAL ANIMALS)
- not examined


GROSS PATHOLOGY (PARENTAL ANIMALS)
- No test item-related abnormal macroscopical findings were noted.


HISTOPATHOLOGY (PARENTAL ANIMALS)
- All histological alterations recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; body weight; food consumption; litter size

2

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; body weight; food consumption; litter size

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

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 AND CLINICAL SIGNS (OFFSPRING)
- At the first litter check, no test item-related abnormal findings were noted. Two pups in the 500 mg/kg/day group in 2 different litters had no milk in the stomach and one pup in the control group had a missing tail tip.
- During the lactation period, no test item-related clinical signs were observed. One litter in the control group had irregular fur growth, which was noted from day 12 post partum onwards. One pup in the 500 mg/kg/day group had a mass on the back but this was not confirmed at necropsy. Another pup had no milk in the stomach and was found dead shortly afterwards.


SEX RATIOS (OFFSPRING)
- The sex ratio was close to 50% in all dose groups (48%, 51%, 51% and 48% males, in order of ascending dose level).


BODY WEIGHT (OFFSPRING)
- Pup body weights in the 500 mg/kg/day group were statistically significantly reduced throughout the lactation period. On day 21 mean body weights were 21% below control in body sexes
- Pup body weights in the 150 mg/kg/day groups were statistically significantly reduced from day 14 (females) and from day 21 (males). On day 21 mean body weights were 7% and 8% below control in males and females respectively.
- In the 50 mg/kg/day group, mean body weight of the pups was similar to that of the control group.


GROSS PATHOLOGY (OFFSPRING)
- No test item-related macroscopical findings were noted at necropsy.

Effect levels (F1)

Dose descriptor:

NOEL

Remarks:

pups

Generation:

F1

Effect level:

50 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; body weight; viability

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The LOAEL in F0 was 150 mg/kg/d based on general toxicity. Reproductive effects occur at 500 mg/kg/d (lower number of implantations, litter size and post-natal loss). Pups weight in the 150 mg/kg/d group was reduces. This effect was attributed to maternal toxicity.

Executive summary:

The study was conducted according to OECD Guideline 415. The purpose of this study was to evaluate any effects of CA2343A (Methyl-Nitro- Oxadiazinamine-Intermediate of Thiamethoxam) on reproductive function as well as neonatal morbidity, mortality, behavior and data on prenatal and postnatal developmental toxicity.

Each group consisted of 24 male and female rats. CA2343A (Methyl-Nitro-Oxadiazinamine- Intermediate of Thiamethoxam) was administered orally, by gavage, once daily throughout a pre-pairing period and up to one day before necropsy at dose levels of:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 50 mg/kg body weight/day

Group 3: 150 mg/kg body weight/day

Group 4: 500 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 (0.5% CMC). All dams and remaining pups were sacrificed on day 21 post partum and males were sacrificed when they were no longer needed for reproduction.

At 500 mg/kg, all males and females were sedated and had ruffled fur at the start of the prepairing period. Food consumption and body weight gain were reduced throughout the prepairing period, resulting in reduced absolute body weight throughout the study. The dams had a reduced number of implantations resulting in fewer fetuses being born per dam. This was considered to be due to parental toxicity as a result of treatment with the test item. The increased post natal loss and the lower body weights of the pups were considered likely to be a result of the parental toxicity.

At 150 mg/kg, all males and females had sedation and ruffled fur for a short period of time during the pre-pairing period. Food consumption and body weight gain were reduced throughout the pre-pairing period, resulting in reduced absolute body weight throughout the study. This was considered to be due to parental toxicity as a result of treatment with the test item. The mean body weight of the pups was reduced, which was considered likely to be a result of the parental toxicity.

At 50 mg/kg, no test item-related clinical signs were noted during the study. Food consumption and body weight were transiently reduced at the start of the pre-pairing period. This was considered to be a result of treatment with the test item but not adverse. No effects on reproduction were noted.

Based on the results of this study, the parental NOAEL was 50 mg/kg/day. The NOEL for effects on reproduction was 150 mg/kg/day. The NOEL for effects on pup growth was 50 mg/kg/day.