Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 24 - October 11, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
There were several deviations from the study protocol during the experiment. All of the deviations were reviewed by the study director, and none were considered to have affected the results of the study.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was mixed with rodent diet and ground.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
This particular strain of rats was chosen as it is susceptible to reproductive toxicants, and the laboratory that performed the study has historical control data for this strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratory, Inc. Raleigh, North Carolina, United States of America
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11-10 weeks old
- Weight at study initiation: (P) Males: 370-474 g; Females: 230-287 g
- Fasting period before study: No
- Housing: Individually in solid-bottomed cages with Bed-O'Cobs bedding. Animals were paired for mating in the male's cage, and females were returned to inidividual cages after the mating period
- Diet: PMI Nutrition Intermational, LLC, Certified Rodent LabDiet 5002 ad libitum (test substance added to diet for exposure groups)
- Water: purified drinking water ad libitum
- Acclimation period: minimum of 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: January 24, 2017 To: April 12, 2017

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: PMI Nutritional International, LLC Certified Rodent LabDiet 5002
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and ground with a mortar and pestle to a fine powder. The basal diet was then added and ground as well. The mixture was placed in a food processor and blended for about 5 minutes. The mixture was then placed in a Hobart mixer with rodent feed, and again mixed for 5 minutes. Additional rodent feed was added to achieve the desired concentration of test substance in the feed. This feed was then placed in a blender and blended for 10 minutes. The feed mixture was then placed in labeled bags.

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PMI Nutritional International, LLC Certified Rodent LabDiet 5002
- Storage temperature of food: room temperature
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Until evidence of mating, or until 14 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- 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:
Doses and homogenity was determined using HPLC. Concentrations of 10,000-50,000 ppm were tested. Samples from the top, middle, and bottom of the formulations were tested. Formulations were also tested after 8 days of storage. Results showed that the analyzed concentrations were within 85%-115% of the target concentration.
Duration of treatment / exposure:
Males: 31 days
Females that became pregnant: 50-58 days
Females that did not become pregnant: 41-54 days
Frequency of treatment:
In diet ad libitum
Details on study schedule:
- Age at mating of the mated animals in the study: 12-13 weeks females; 14-15 weeks males
Doses / concentrationsopen allclose all
Dose / conc.:
10 000 ppm
Remarks:
equivalent to 1200 mg/kg bw/day based on a conversion factor of 0.12 for rats by EFSA
Dose / conc.:
30 000 ppm
Remarks:
equivalent to 3600 mg/kg bw/day based on a conversion factor of 0.12 for rats by EFSA
Dose / conc.:
50 000 ppm
Remarks:
equivalent to 6000 mg/kg bw/day based on a conversion factor of 0.12 for rats by EFSA
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dosage based on previous subchronic and chronic toxicity studies which showed no toxic effects at 0, 1, 3, and 5% of diet.
- Rationale for animal assignment: random
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked: moribundity and mortality all animals, dystocia or other labor difficulties for pregnant females at or near parturition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly all animals; gestation days 0, 4, 7, 11, 14, 17, and 20, lactation days 1, 4, 7, 10, and 13 for pregnant females

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Oestrous cyclicity (parental animals):
4-6 days
Sperm parameters (parental animals):
Parameters examined in P male parental generations: seminal vesicles, testes with epididymides, vas deferens
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/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, anogenital distance (AGD), presence of nipples/areolae in male pups,

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 at 31 days after study initiation
- Maternal animals: All surviving animals, lactation day 13

GROSS NECROPSY
- Gross necropsy consisted of external surface, orifices, cranial cavity, external surface of the brain, thoraic, cavity, abdominal cavity , and pelvic cavity

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate gland, seminal vesicle, spleen, testes, thymus gland, thyroids
Histopathology: brain, coagulating glands, kidneys, liver, ovaries, oviducts, pituitary gland, prostate gland, seminal vesicles, mammary glands, testes with epididymides, vas deferens, uterus, cervix, vagina, gross lesions
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: gross necropsies and thyroids were collected for histopathological examination - 1 pup per litter

GROSS NECROPSY
- Gross necropsy consisted of examined viscerally, stomach contents were also noted, abdominal cavity, and urinary/reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroids were prepared for microscopic examination.
Statistics:
Standard deviation, standard error, two-tailed tests with minimum significance levels of 1% and 5%.

For parental mating, fertility, conception, and copulation indices were analyzed using Chi-square tests with Yates' correction factor.

For body weights, body weight changes, food consumption, estrous cycle length, precoital interval, gestation length, number of former implantation sites, unaccounted-for sites, number of pups born, live litter size, absolute organ weights, relative organ weights, thyroid hormone values, anogenital distance, and number of nipples/areolae, parametric one-way ANOVA was used to determine intergroup differences. If significant intergroup variance was found, Dunnett's test was used to compare control to test groups.

For proportion of males at birth, and postnatal survival, the Kruskal-Wallis non-parametric ANOVA test was used. If significant intergroup variance was found, Dunnett's test was used to compare control to test groups.
Reproductive indices:
Parental mating
Fertility
Conception
Copulation indices
Gestation length
Number of implantation sites
Offspring viability indices:
Mean litter size
Postnatal survival

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hair loss and red material on body surfaces was noted for some treatment group animals. However, these findings were infrequent and occurred at similar frequencies as the control group. Thus, these observations were not considered to be treatment related.
Mortality:
no mortality observed
Description (incidence):
All parental animals survived to scheduled necropsy.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Significantly higher food consumption was seen in lactating females on Days 10-13 of lactation in the 30,000 and 50,000 ppm groups. As the effect was transient and did not affect mean body weights, it was not considered treatment related. Test substance consumed was higher in lactating females, but this is a common effect seen in nursing animals and was not considered treatment related.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis of males showed no treatment related effects.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
One male animal in the 50,000 ppm group showed minimal focal sperm stasis in the left testes. As this male sired a litter, and this is a periodic finding in rats, this observation was not considered to be treatment related. Other incidental tissue alterations were noted, but as there was no increase in severity, prevalance, or histologic character, these were not considered to be treatment related.
Histopathological findings: neoplastic:
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

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
50 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to the highest dose of 50,000 ppm (equivalent to 6000 mg/kg bw/day based on a conversion factor of 0.12 for rats by EFSA).

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Some pups in each group, including the control and treatment groups were found dead or euthanized in extremis. Some pups in all groups except the 10,000 ppm group were missing and presumed cannibalized. There were no significant differences in mortality between the control and treatment groups, and thus the deaths were not thought to be treatment related.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis showed no difference between treatment and control groups.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One pup in the 50,000 ppm group showed a dilated renal pelvis, and another in the 50,000 ppm group showed scabbing in the dorsal thoracic area. Neither finding was considered to be related to treament of the parents.
Histopathological findings:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Higher anogenital distances were noted in both male and female pups in litters from the 10,000 ppm dose group. This was not considered to be treatment related as the effect was not seen in higher dose groups, and values were within historical controls. Number of areolae/nipples in male pups was not stastically higher in pups from treatment group parents than in pups from control group parents.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
50 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to the highest dose of 50,000 ppm (equivalent to 6000 mg/kg bw/day based on a conversion factor of 0.12 for rats by EFSA).

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Reproductive Performance

Parameter

0 ppm

10,000 ppm

30,000 ppm

50,000 ppm

Male Mating Index (%)

90.0

100.0

100.0

90.0

Female Mating Index (%)

90.0

100.0

100.0

90.0

Male Fertility Index (%)

90.0

90.0

100.0

80.0

Female Fertility Index (%)

90.0

90.0

100.0

80.0

Male Copulation Index (%)

100.0

90.0

100.0

88.9

Female Conception Index (%)

100.0

90.0

100.0

88.9

Estrous Cycle Length (Days)

5.0

4.4

5.4

4.0

Pre-Coital Interval (Days)

2.9

2.6

2.4

1.9

 

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive and developmental toxicity is 50,000 ppm (equivalent to 6000 mg/kg bw/day based on a conversion factor of 0.12 for rats by EFSA) in the diet based on the absence of treatment related effects in parental animals and the offspring.
Executive summary:

The reproductive/developmental toxicity of the test substance was determined in an OECD Guideline 421 test. Groups of 10 male and 10 female rats were fed diets with 0, 10,000, 30,000, or 50,000 ppm of the test substance in the diet for 14 days prior to mating and during mating. The females continued to be fed the test substance through gestation and lactation. The resulting litters were examined for viability and mortality. No treatment related adverse effects from the test substance were seen in any parental animal. No treament related adverse effects were seen in the offspring. The NOAEL for the test substance for reproductive and developmental toxicity was 50,000 ppm (equivalent to 6000 mg/kg bw/day based on a conversion factor for rats according to the "Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data" (EFSA, 2012)).

Reference:

EFSA (2012) Guidance on selected default values to be used by the EFSA Scientific Committee, Scientific Panels and Units in the absence of actual measured data. EFSA Journal,10 (3), 2579.