Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer-reviewed journal.

Data source

Reference
Reference Type:
publication
Title:
Oral developmental toxicity study of test chemical in rats
Author:
B.A. Magnuson et.al.
Year:
2007
Bibliographic source:
Food and Chemical Toxicology 45 (2007)

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: US Food and Drug Administration principles (FDA, 1982) and Organization for Economic Co-operation and Development (OECD) guidelines for developmental toxicity (No. 414) (OECD, 2006).
Principles of method if other than guideline:
The present study was undertaken to determine the reproductive toxicity potential of test chemical.when administered orally to rats during the period of major organogenesis and histogenesis.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methylsulfonylmethane (MSM)
- Molecular formula (if other than submission substance): C2-H6-O2-S
- Molecular weight (if other than submission substance): 94.1334 g/mole
- Substance type: Organic
- Physical state: Solid
- Impurities (identity and concentrations): Purity: 99.9%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Cardinal Nutrition, Inc. (Vancouver, WA).
- Expiration date of the lot/batch: No data
- Purity: 99.9%

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature.
- Stability under test conditions: the formulations were stable for 32 days for concentrations ranging from 10 to 100 mg/ml when stored at room temperature.
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No data
- Preliminary purification step (if any): No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data

FORM AS APPLIED IN THE TEST (if different from that of starting material): No data

OTHER SPECIFICS: No data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: between 145 and 215 g/rat
- Fasting period before study: No data available
- Housing: Quarantine in plastic cages
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): Harlan Teklad Certified Rodent Diet #8728C, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 4 degC
- Humidity (%): No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were prepared in deionized water at 10, 100, and 200 mg/ml concentrations for the 50, 500, and 1000 mg/kg dose levels, respectively.

DIET PREPARATION
- Rate of preparation of diet (frequency):No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: 50, 500,1000 mg/kg
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): No data
- Purity: No data
Details on mating procedure:
- M/F ratio per cage: 1:1 mating scheme (i.e., one male:one female)
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No data available
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): males were removed and females were individually housed for the rest of the experiment.
- Any other deviations from standard protocol: To facilitate the teratogenic evaluation of pups, a staggered-start design (via staggered mating) was employed. Mating was staggered over six days resulting in six series.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Aliquots of the formulations were transferred to dosing vials and samples representing each dose level from the first preparation were analyzed by gas chromatography. The purity of test chemical was 99.9%.
Duration of treatment / exposure:
14 days (on gestation days 6–20)
Total exposure period: 20 days
Frequency of treatment:
Daily
Details on study schedule:
Terminal killing: 21st day of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: mg/kg
Remarks:
vehicle group
Dose / conc.:
50 other: mg/kg
Dose / conc.:
500 other: mg/kg
Dose / conc.:
1 000 other: mg/kg
No. of animals per sex per dose:
Total: 98
0 mg/kg/day : 25 timed-bred primiparous dams
50 mg/kg/day : 24 timed-bred primiparous dams
500 mg/kg/day : 25 timed-bred primiparous dams
1000 mg/kg/day : 24 timed-bred primiparous dams
Control animals:
yes
Details on study design:
No data available
Positive control:
No data available

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on gestation days 0 (sperm-positive day/randomization), 3, 6, 9, 12, 15, 18, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Feed consumption was calculated for each period between weighings.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:
Oestrous cyclicity (parental animals):
No data available
Sperm parameters (parental animals):
No data available
Litter observations:
No data available
Postmortem examinations (parental animals):
SACRIFICE
All dams were euthanized using CO2 asphyxiation on their 21st day of gestation and underwent a cesarean section.

GROSS NECROPSY
Gross necropsy consisted of examination of the brain and all organs in the thoracic and abdominal cavities of the dams.
Pregnancy status, number of corpora lutea was examined.
The uterus and ovaries were weighed.

Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of external morphological and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:
Dam weights, litter body weights, and viability data were analyzed by a two-way analysis of variance (ANOVA). In the presence of a significant main effect, all post-hoc comparisons were performed using Dunnett’s test (two-tail). Gross, visceral, cephalic, and skeletal data were analyzed by Chi-Square/Fisher’s Exact tests (fetal N) when incidence in the treated rats was higher (i.e., when the difference in the absolute number of fetuses affected is greater than three) than controls. A minimum statistical significance level of p ≤ 0.05 was used in all cases.
Reproductive indices:
No data available
Offspring viability indices:
No data available

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed.
Dermal irritation (if dermal study):
not examined
Mortality:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight was similar among all groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption was not affected.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Uterine weights were similar across all groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross external anomalies were observed.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histopathological lesions in organ and tissues were observed.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
No evidence of maternal toxicity was observed.

Details on results (P0)

No evidence of maternal toxicity was observed.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive performance
Remarks on result:
other: No evidence of maternal toxicity was observed.

Target system / organ toxicity (P0)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were 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):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Oral administration of test chemical to pregnant rats at doses of 50, 500, or 1000 mg/kg/day did not result in any structural malformations or fetal anomalies as evaluated by gross external, cephalic, visceral and skeletal examinations.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not specified

Details on results (F1)

No significant differences in litter viability, litter size, or litter body weight were detected. Fetal evaluations failed to show any biologically significant increase in the incidence of anomalies in the treated groups, and no malformations were seen in any of the fetuses. No evidence of fetal mortality, alterations to growth, or structural alterations were observed in the fetuses of dams administered 50–1000 mg/kg/day.

Embryo-fetal toxicity summary
No evidence of embryo or fetal toxicity, or treatment related alterations in fetal body weights or fetal examinations (gross external, visceral, cephalic, or skeletal) was observed in this study at doses up to 1000 mg/kg .

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
other: Fetotoxicity
Remarks on result:
other: No evidence of embryo-fetal toxicity was observed.

Target system / organ toxicity (F1)

Critical effects observed:
not specified
System:
other: not specified
Organ:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified
Treatment related:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity was considered to be 1000 mg/kg/day.When female rats were treated with test chemical orally.
Executive summary:

The objective of the study was to determine the reproductive toxicity potential of test chemical when administered orally to pregnant rats during the period of major organogenesis and histogenesis. In a preliminary dose-finding study, distilled microprill (i.e., microspherical pellets of test chemical ) was administered by oral gavage at dose levels of 0 (vehicle control), 50, 250, 500, and 1000 mg/kg/day to 8–9 sperm-positive female Sprague–Dawley rats/group/day on gestation days 6–20. No evidence of maternal or fetal toxicity was observed. For the definitive study, four groups of 24–25 timedbred primiparous female rats were administered 0, 50, 500, or 1000 mg /kg/day via gavage on gestation days 6–20. Maternal feed consumption, body weight, body weight gain, uterus weight and corrected body weight/body weight gain were unaffected by treatment. No evidence of maternal toxicity, and no significant differences in litter viability, litter size, or litter body weight were detected. Fetal evaluations failed to show any biologically significant increase in the incidence of anomalies in the treated groups, and no malformations were seen in any of the fetuses. No evidence of fetal mortality, alterations to growth, or structural alterations were observed in the fetuses of dams administered 50–1000 mg/kg/day. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity was considered to be 1000 mg/kg/day.When female rats were treated with test chemical orally.