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:

2008-03-11 until 2009-03-27

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:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

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:

Hill formula: C4H8N4O3 CAS 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 Thiamethoxam)
- Substance type: mono-constituent organic substance
- Physical state: White fine crystals
- Analytical purity: 99.3 %
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: 427801
- Expiration date of the lot/batch: 31 July 2010
- Stability under test conditions: 7 days in 0.5% CMC (vehicle) 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, Füllinsdorf / Switzerland
- Age at study initiation: P: Males: 8 weeks, females: 9 weeks
- Weight at study initiation: (P) Males: 236 to 279 g; Females: 163 to 203 g
- Fasting period before study: not reported
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- 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 Füllinsdorf was available ad libitum in water bottles.
- Acclimation period: 7 d. Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


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-hour fluorescent light / 12-hour dark cycle with music during the light period


IN-LIFE DATES: From: 2008-03-18 To: 2008-08-01

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): The dose formulations were prepared weekly and split into 7 daily aliquots for dosing.
- Mixing appropriate amounts with (Type of food): Test item 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.
- Storage temperature of food: Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.


VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% CMC aqueous solution; no justification given
- Concentration in vehicle: 5, 15, and 50 mg/ml
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no. (if required): 1367388 34707017
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 21 d
- Proof of pregnancy: vaginal plug / sperm in vaginal smear, referred to as day 0 post coitum (of pregnancy)
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. not applicable
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding. All dams were allowed to give birth and rear their litters (F1 pups) up to day 21 post partum.
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

This analytical part was conducted at Harlan Laboratories Ltd, Itingen, Switzerland under GLP-compliant conditions to verify the identity of the test item CA2343A administered and todetermine its content, homogeneity and stability of dose preparations.
Several dose preparations were prepared and representative analytical samples were collected and dispatched to the analytical laboratories internally. The test item concentrations were determined by HPLC coupled to a UV detector and quantified with the area under the peak.
The identity of CA2343A was confirmed by its retention time which was similar to that measured in the working standards. The content of 11 out of a total of 12 samples met the required content limit of ±10% with reference to the nominal concentration. In addition, the homogeneous distribution of CA2343A in 0.5% carboxymethylcellulose aqueous solution was demonstrated. The dose preparations were considered to be stable 7 days when kept at room temperature.
In conclusion, the results obtained within this part confirm the correct preparation and storage of dose preparations during the conduct of this study.

Duration of treatment / exposure:

Males: minimum 11 weeks
Females: approximately 11 weeks

Frequency of treatment:

once daily

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 - 19 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg bw/d
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

24 male per group and 24 female per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of previous studies available in Sponsor’s files.
- Rationale for animal assignment (if not random): 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.
- Other:

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in tables are included in the original study report


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily


BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.


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
- 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): Not applicable
- Time schedule for examinations:


OTHER:

Oestrous cyclicity (parental animals):

Observations recorded on:
- Ovaries: congestion
- Uterus: implanatation site, atrophy
- Vagina: anestrus, proestrus, estrus, metestrus, diestrus, mucosal atrophy , mucosal mucification
- Pituary gland: stomatodeal rests, Cystic Rathke's cl., Cyst:pars anterior, hypertrophy (focal)
- Skin / subcutis: no histological cor., epithelial hyperpl.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
- Testes: tubular degeneration
- Epididymides: cellular detritus, aspermia, epithelial vacuol.
- Prostate gland: inflammation

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (1:1 ratio/sex/litter as nearly as 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:
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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals. P generation males were sacrificed when they were no longer needed for the assessment of reproductive effects.
- Maternal animals: all surviving animals. At or shortly after weaning, P generation females and the remaining pups were sacrificed.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively.
Gross lesions
Ovaries
Pituitary
Prostate
Seminal vesicles with coagulating gland
Testes with epididymides (in Bouin’s fixative)
Uterus and cervix
Vagina
Target organs

Postmortem examinations (offspring):

Not applicable since one generation study

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:

Females: Percentage mating, Fertility index (%), Conception rate (%), Gestation index, birth index, weaning index
Males: Mating performance

Offspring viability indices:

Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) * 100

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:

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:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All males and females in the 500 mg/kg/day group had sedation on the first day of dosing only and ruffled fur for up to 2 weeks at the start of the pre-pairing period. At 150 mg/kg/day, all males and females had sedation on the first day of treatment only and ruffled fur for up to 5 days at the start of the study.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption:
At 500 mg/kg/day group, mean food consumption was statistically significantly reduced over the first few weeks of the pre-pairing period in both males and females, after which it was similar to control. Food consumption was reduced in the dams during the last part of the lactation period. At 150 mg/kg/day, mean food consumption was statistically significantly reduced at the start of the pre-pairing period and slightly lower than control (not statistically significant) at the end of the lactation period. At 50 mg/kg/day, there was a transient reduction in mean food consumption in males during the first week of the pre-pairing period. The food consumption of the females was unaffected by treatment with the test item.

Body weight:
At 500 mg/kg/day, mean body weight was reduced throughout the study in both males and females. The maximum differences in body weight from control were 13% and 17% in males and females respectively. Mean body weight gain was statistically significantly reduced in both sexes during the pre-pairing period and in the dams during the gestation period. At 150 mg/kg/day, mean body weight was statistically significantly reduced during the majority of the study in both sexes. Mean body weight gain was statistically significantly in both sexes during the pre-pairing period. At 50 mg/kg/day, there was a transient reduction in body weight and body weight gain in males at the start of dosing. Thereafter weights and weight gains were similar to those of the control group. In the females, body weights and body weight gains were not affected by treatment with the test item.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Test substance generally well-tolerated. No test item-related abnormal macroscopic or histopathological findings were noted, all animals survived until scheduled necropsy.


REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
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) and the mean number of pups at the first litter check was also statistically significantly reduced. Post-natal loss was statistically significantly increased (this increase was partly due to one dam, which lost all 12 pups.


REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No unusual findings reported.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No unusual findings reported.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No unusual findings reported.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No unusual findings reported.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No unusual findings reported.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Parental toxicity: At 150 and 500 mg/kg bw/d, sedation and ruffled fur in males and females. Food consumption and body weight gain reduced in the pre-pairing period, resulting in reduced absolute body weight.

2

Dose descriptor:

LOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Parental toxicity: see above

3

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Effects on reproduction: At 500 mg/kg bw/d increased post natal loss and lower body weights of the pups. These results were considered to be due to parental toxicity following treatment with the test item.

4

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Effects on reproduction: see above

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

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)
At 500 mg/ kg bw/d (dose of P animals) the mean number of pups at the first litter check was also statistically significantly reduced. Post-natal loss was statistically significantly increased (this increase was partly due to one dam, which lost all 12 pups.


CLINICAL SIGNS (OFFSPRING)
No abnormal observations reported.


BODY WEIGHT (OFFSPRING)
Pup weights in the 500 mg/kg/day groups were statistically significantly reduced throughout the lactation period. On day 21 mean body weights were 21% below control in both sexes. Pup weights in the 150 mg/kg/day group were statistically significantly reduced from day 14 (females) and from on day 21 (males). On day 21 mean body weights were 7% and 8% below control in males and females respectively.


SEXUAL MATURATION (OFFSPRING)
The sex ratio was close to 50% in all dose groups.


ORGAN WEIGHTS (OFFSPRING)
Not examined (bodies preserved after sacrifice)


GROSS PATHOLOGY (OFFSPRING)
At the first litter check, no test item-related abnormal findings were noted. During the lactation period, no test item-related clinical signs were observed.


HISTOPATHOLOGY (OFFSPRING)
Not examined (bodies preserved after sacrifice)


OTHER FINDINGS (OFFSPRING)

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Effects on pup growth: At 150 mg/kg bw/d (dose of P animals) reduced body weight of pups. These results were considered to be due to parental toxicity following treatment with the test item.

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Effects on pup growth: see above

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the parental NOAEL (No Observed Adverse Effect Level) was 50 mg/kg/day. The NOEL (No Observed Effect Level) for effects on reproduction was 150 mg/kg/day. The NOEL (No Observed Effect Level) for effects on pup growth was 50 mg/kg/day. A classification of the test item applies as to its reprotoxic properties is not necessary.

Executive summary:

The purpose of this oral toxicity study was to provide general information concerning the effects of CA2343A (Methyl-Nitro-Oxadiazinamine-Intermediate of Thiamethoxam) on reproductive function as assessed by gonadal function, estrus cycles, mating behavior, conception, parturition, lactation, weaning, and the growth and development of the offspring. The study also provided information about the effects of CA2343A (Methyl-Nitro- Oxadiazinamine-Intermediate of Thiamethoxam) on neonatal morbidity, mortality, behavior and data on prenatal and postnatal developmental toxicity as well as serving as a guide for subsequent tests.

CA2343A (Methyl-Nitro-Oxadiazinamine-Intermediate of Thiamethoxam) was administered orally by gavage once daily to males over a pre-pairing and pairing period up to one day before necropsy and to females over the pre-pairing, pairing, gestation and lactation period up to day 20 post partum at dose levels of 0, 50, 150 and 500 mg/kg/day.

At 150 and 500 mg/kg, sedation and ruffled fur were noted in the males and females. Food consumption and body weight gain were reduced in the pre-pairing period, resulting in reduced absolute body weight. At 500 mg/kg/day, there was increased post natal loss and the lower body weights of the pups. At 150 mg/kg/day, the body weight of the pups was reduced. These results were considered to be due to parental toxicity following treatment with the test item. 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 (No Observed Adverse Effect Level) was 50 mg/kg/day. The NOEL (No Observed Effect Level) for effects on reproduction was 150 mg/kg/day. The NOEL (No Observed Effect Level) for effects on pup growth was 50 mg/kg/day. A classification of the test item applies as to its reprotoxic properties is not necessary.