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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2011-March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD and US EPA guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: US EPA OPPTS 870.3550
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD guideline 421 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: light brown, clear, viscous liquid
- Storage condition of test material: stored at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI or Raleigh, NC
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 331-391 g; Females: 211-291 g
- Fasting period before study: no
- Housing: until pairing: individually in stainless steel wire-mesh cage; paired for mating in male cage; after mating males were housed in suspended wire-mesh cages; females were transferred to plastic maternity cages with nesting material, ground corncob beding.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: ad libitum PMI Nutrition International, LLC Certified Rodent LabDiet 5002
- Water: ad libitum (reverse-osmosis-purified on-site drinking water)
- Acclimation period: minimum of 10 days for males and 15 days for females

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2-21.4
- Humidity (%): 42.9-48.8
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 2 December 2011 To: 22 January 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance formulations were prepared approximately weekly for each dosage level, divided into daily aliquots and stored refrigerated. Test substance concentrations were 0, 4, 8, 16 and 32 mg/mL.
Dosage volume for all groups was 5 mL/kg.


VEHICLE
- Lot/batch no.: 2AD0465
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected from the middle stratum of each dosing formulation (including control group) prepared during the first and the last week of dose administration. Analyses were conducted by means of a validated gas chromatography method using flame ionization detection. Mean concentrations ranged from 95.3% to 108% of target concentration.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or the presence of sperm following a vaginal lavage referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually into plastic maternity cages with nesting material, ground corncob bedding.
Duration of treatment / exposure:
males: 14 daily doses prior to mating; throughout mating period for a total of 31 doses
females: 14 daily doses prior to pairing, dosed through lactation day 3 (females that delivered) or post-mating day 25 (females that failed to deliver) for a total of 39-51 doses
Frequency of treatment:
daily
Duration of test:
until lactation day 4
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 40, 80, 160 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of the 28-day OECD 407 study, where a NOAEL of 150 mg/kg bw/day was observed (no significant effects observed). For the present screening study a top dose of 160 mg/kg bw/day was included to increase the probabilty for detecting some adverse effects, without risking the possibility of excess toxicity due to the longer duration.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- for moribundity and mortality, and for signs of toxicity 2 hours following dose administration. Females expected to deliver were observed for dystocia or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for males and weekly for females until evidence of copulation. Female body weights were recorded on GD 0, 4, 7, 11, 14, 17 and 20 and on lactation days 0, 1 and 4.
FOOD CONSUMPTION
- Food consumption for each animal determined as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day #4
- Organs examined: according to guidelines
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No (dams delivered)
- Number of late resorptions: No (dams delivered)
Fetal examinations:
- External examinations: Yes, Fo animals delivered and the dams and her litter were euthanized on LD/PND 4. All pups were examined.
Statistics:
Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
A parametric one-way ANOVA to determine intergroup differences was applied to mean parental BW, BW changes and food consumption, offspring BW and BW changes, gestation length, numbers of implantation sites, number if pups born, live litter size on PND 0, corpora lutea, organ weights (absolute and relative) and pre-coital intervals. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Kruskal-Wallis nonparametric ANOVA was used to determine intergroup differences for mean litter proportions of males at birth and postnatal survival. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn's test was used to compare the test substance-treated groups to the control group. Histopathological findings of treated groups were compared to the control group by the Fisher's Exact test.
Indices:
Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0/ No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-selection) (% Per Litter) =
(Sum of (Viable Pups Per Litter on PND 0 or PND 4 [Pre-selection]/No. of Pups Born Per Litter)/No. of Litters Per Group) x 100

Postnatal Survival for All
Other Intervals (% Per Litter) = (Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/No. of Litters Per Group) x 100

Where N= PND 0-1 and 1-4

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In high dose females a statistically significant increased relative liver weight (m/f) and increased absolute liver weight (f) were observed. These effects are considered non-adverse.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
>= 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Treatment with test item by oral gavage in male and female Sprague-Dawley rats at dose levels of 20, 40, 80 and 160 mg/kg bw/day according to EU/EPA test guidelines did not result in adverse effects in the dams. Based on the absence of adverse effects in the dams, the NOAEL for maternal systemic toxicity is at least 160 mg/kg bw/day. No reproduction and developmental toxicity was observed for treatment up to 160 mg/kg bw/day. A reproduction and developmental NOAEL of at least 160 mg/kg bw/day was derived.
Executive summary:

Test Guidance

According to OECD 421 and US EPA OPPTS 870.3550 guidelines

Method and materials

Test item was administrated by daily oral gavage to male and female Sprague-Dawley rats at dose levels of 20, 40, 80 and 160 mg/kg bw/day . Males were exposed for 2 weeks prior to mating, during mating and up to termination (total of 31 doses). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and during 3 days of lactation (39 -51 doses).

Results

Analysis demonstrated mean concentration ranges from 95.3% to 108% of target concentrations.

Parental toxicity was observed at the high dose only, and consisted of slightly lower body weight gain in high dose male rats at 160 mg/kg bw/day. The observed statistically significant increased relative liver weights in males and females, and increased absolute liver weights in females, together with increased hepatocellular vacuolation and increased minimal tubular dilation in kidneys observed in high dose males are considered non-adverse. Based on these observations in high dose animals, the NOAEL for parental systemic toxicity is 80 mg/kg bw/day. Maternal toxicity was at least 160 mg/kg bw/day, as no adverse effects were observed in dams.

Conclusions

No reproduction and developmental toxicity was observed for treatment up to 160 mg/kg bw/day. A reproduction and developmental NOAEL of at least 160 mg/kg bw/day was derived.