Benzenamine, N,N-dimethyl-, oxidized, molybdatetungstatephosphates

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Aug 2017 - 22 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, N,N-dimethyl-, oxidized, molybdatetungstatephosphates

EC Number:

309-916-8

EC Name:

Benzenamine, N,N-dimethyl-, oxidized, molybdatetungstatephosphates

Cas Number:

101357-19-1

Molecular formula:

Reaction mass of C25 H30 N3. P Moy Wz O40 (y +z = 12) , C24 H28 N3. P Moy Wz O40 (y +z = 12) and C23 H26 N3. P Moy Wz O40 (y +z = 12)

IUPAC Name:

Reaction mass of [4-[bis[4-(dimethylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]-dimethyl-ammonium. molybdatetungstatephosphates, [4-[[4-(dimethylamino)phenyl]-[4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]-dimethyl-ammonium. molybdatetungstatephosphates and [4-[bis[4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]-dimethyl-ammonium. molybdatetungstatephosphates

Test material form:

solid: nanoform, no surface treatment

Details on test material:

SOURCE OF TEST MATERIAL
-Product number: 0345N (skin sensitisation, OECD 422, eye irritation, acute oral toxicity)
0345F (Ames test)

- Purity Preparation containing ≥80% (treat as 100%)
- Physical form/colour: Violet powder
- Expiry date: Stable for at least 5 years from delivery date

-Shape Category: spheroidal
-Shape: spherycal
-pure shape: yes
-Typical composition: ca. 100%
-range >50 and < 100%

-D10, typical value: 30 nm, range: >1.5 and < 50nm
-D50, typical value: 45 nm, range: >15 and <100nm
-D90, typical value: 60nm, range: >25 and < 150nm

-Fraction in size range 1-100nm: > 50 and <100%

-Crystallinity:
Structure:
amorphous, pure structure

-SSA: BET surface area typically ca. 32-40 m2/g for product number 345F and 0345N)
Range > 10 and < 200 m2/g

-.no surface treatment

Storage conditions: Stored at ambient conditions in the dark (away from direct light)
- Safety precautions: Routine hygienic procedures (nitrile gloves, goggles, face mask)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 982432
- Expiration date of the lot/batch:
- Purity Preparation containing ≥80% UVCB (treat as 100%)
- Physical form/colour: Violet powder
- Manufacture/synthesis date: 21-Apr-1998
- Expiry date: Stable for at least 5 years from delivery date
- Storage conditions: Stored at ambient conditions in the dark (away from direct light)
- Safety precautions: Routine hygienic procedures (nitrile gloves, goggles, face mask)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 331-431 g Females: 179-245 g
- Housing: Cages with standard, granulated, S8-15 sawdust bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30 - 70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/ 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

VEHICLE
- Batch number KMO9422, KM09047
- Expiry date: August 2018
- Storage conditions: Stored at ambient conditions
- Safety precautions: Routine hygienic procedures (nitrile gloves, goggles, face mask)

- Concentration in vehicle: 20 mg/mL to 200 mg/mL
- Administration volume: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 1-4 days
- Proof of pregnancy: Ejected copulation plugs. Sperm within vaginal smear.
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Validated analytical method in place before commencement of treatment.
Formulations at two concentration levels (target concentrations: 200 and 20 mg/mL) were prepared and the following parameters were determined:
• System suitability (SST)
• Linearity
• Accuracy/repeatability
• Homogeneity
• Specificity
• Limit of Quantification (LOQ) and Limit of Detection (LOD)
• Stability [autosampler, formulations (20 ± 5 ºC, in the absence of light, for 7-12 days)]

The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify their correct preparation.
Control (vehicle) formulations were analyzed to confirm the absence of test item or other substances at the retention time of the test item.

Results are expected to be within ±20% of nominal value and the coefficient of variation (CV) should be ≤10%.
Other parameters were also tested, to check whether they met the following acceptance criteria:
• System Suitability Test (SST) --- CV ≤ 2%.
• Calibration curve --- R2 ≥ 0.99; deviation of the calibration standards ≤ 10% (75% should comply).

Duration of treatment / exposure:

5 - 8 weeks

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Doses selected based on preliminary results obtained from 14-Day non GLP Dose range finder study
- Rationale for animal assignment (if not random): Random
- Other:

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1 and weekly thereafter

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week

Oestrous cyclicity (parental animals):

Dry smears Using inoculation loops during the following phases:
• For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
• Daily from the beginning of treatment period until evidence of mating.
• On the day of necropsy
A cotton swab impregnated in physiological saline was used in order to check the evidence of mating during the mating period.

Sperm parameters (parental animals):

At necropsy miicroscopic examination of the epididymus, prostate and seminal vesicles with coagulating glnads and testes were examined , seminiferous tubules were also evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages.

Litter observations:

Clinical observations Observed 24 hours after the considered birth and then daily for evidence of ill-health or reaction to maternal treatment
Litter size Daily from Day 1-13 of age
Sex ratio Days 1, 4, 7 and 13 of age
Individual offspring body weights
Days 1, 4, 7 and 13 of age
Ano-genital distance Day 1 - all F1 offspring
Nipple/areolae count Day 13 of age - male offspring.

Postmortem examinations (parental animals):

SACRIFICE
F0 Males After final investigations completed (after 5 weeks of treatment)
F0 Females failing to produce viable litter (not pregnant)

Day 25-26 after mating
F0 Females whose litters die before Day 13
On or after day last offspring dies
Females killed at termination
Day 14-16 of lactation
F1 Offspring Selected offspring for thyroid hormone analysis – Day 4 of age (two females per litter where possible)
Scheduled sacrifice - Day 13-15 of age


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Complete necropsy performed for F0 animals (five males and five lactating females with a surviving litter per group). Number of uterine implantation sites counted and checked.

HISTOPATHOLOGY / ORGAN WEIGHTS
All tissues from the animals above were prepared for microscopic examination and weighed, respectively.
Routine staining 4-5 um sections stained with hematoxylin and eosin, except testes which are also stained with periodic acid-Schiff
Extension of initial examination Liver from five males in Group 2 and five males in Group 3 selected at scheduled euthanasia.

Postmortem examinations (offspring):

F1 offspring on Day 4 of age Blood sampling taken from two females per litter where possible.
External macroscopic examination.
F1 offspring
on Day 13 of age Blood sampling taken from two males and two females per litter, where possible.
Thyroid gland was retained from one male and one female in each litter where possible.
All animals were subjected to an external macroscopic examination; particular attention was paid to the external genitalia.

Statistics:

Statistical analysis was performed on the following data types at each time point separately:

- Body weight, using absolute weights
- Food consumption, over appropriate study periods
- Blood chemistry and hematology (including coagulation)
- Hormone analyses
- Organ weights, both absolute and adjusted for terminal body weight
- Grip strength and motor activity
- Estrous cycles pre-coital interval
- Mating performance and fertility
- Gestation length
- Litter data (litter size, offspring survival, offspring body weight) ano-genital distance, average for each litter adjusted for litter average pup body weight
- Number of nipples/areolae in male pups counted on post natal Day 13

Reproductive indices:

- Grip strength and motor activity
- Estrous cycles pre-coital interval
- Mating performance and fertility
- Gestation length
- Litter data (litter size, offspring survival, offspring body weight) ano-genital distance, average for each litter adjusted for litter average pup body weight
- Number of nipples/areolae in male pups counted on post natal Day 13

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

water consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

reproductive function (oestrous cycle)

reproductive function (sperm measures)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

food efficiency

water consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The No Observed Effect Level (NOEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring on litter size, survival, sex ratio, clinical signs, body weights, anogenital distances or macropathology.