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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats according to OECD Guideline 422 a NOEL of 1000 mg/kg bw/d for reproduction/development was determined, 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, ano-genital distances or macropathology.

Link to relevant study records

Reference

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

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

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)

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

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:

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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)

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening (OECD 422), three groups of 12 male and 12 female rats received Pigment Violet 3 PTM at the doses of 100, 300 and 1000 mg/kg/day, respectively. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 32 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 14-16 of lactation (the day before sacrifice). Females were allowed to litter and rear their offspring, and litters were killed on Day 13-15 of lactation (the day before the corresponding female was killed). F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, arachis oil.

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology, coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated. Clinical signs, behavior assessment, litter size, survival, sex ratio, body weight and macropathology were also assessed for all offspring.

The study results can be summarized as follows:

• There were no deaths considered related to treatment with Pigment Violet 3 PTM.
• Administration of Pigment Violet 3 PTM at 100, 300 or 1000 mg/kg/day was considered not to have any effects on clinical condition, body weight, food consumption, motor activity, sensory reactivity and grip strength, pre-coital interval, mating performance and fertility, or gestation length. Violet feces due to the test item color were recorded during treatment in all test-item-administered groups. Salivation was recorded in all test-item-administered groups; the incidence was dose-related in both sexes and is most likely due to taste aversion (from gavage dosing).
• All females allocated to the study showed regular 4-day estrous cycles before and during treatment. At termination, all reproductive phase females showed diestrus with the exception of 2/12 animals at 100 mg/kg/day, which had recovered estrous cycle.
• There were no differences in hematology, coagulation, clinical biochemistry or organ weights considered treatment-related. T4 analyses of samples in Main study males and F1 offspring on day 13 did not reveal any differences that could be attributable to treatment.
• There were no macroscopic findings that could be considered test-item-related.
• Histopathology reveals that there were no treatment-related effects in any of the examined organs or in the reproductive organs or mammary glands.
• There was no effect on offspring survival due to Pigment Violet 3 PTM or on litter size, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

In conclusion, the effects of oral (gavage) administration of Pigment Violet 3 PTM to Wistar rats receiving 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Systemic toxicity:

The No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.

Reproductive / developmental toxicity:

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, ano-genital distances or macropathology.

Effects on developmental toxicity

Description of key information

In an oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats according to OECD Guideline 422 a NOEL of 1000 mg/kg bw/d for reproduction/development was determined, 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, ano-genital distances or macropathology.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

As a result the substance is not classified for reproductive or developmental toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.