Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted Jan. 2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted August 1998
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Laromer LR 8887
- Physical state: Clear light yellowish liquid
- Analytical purity: 78%
- Lot/batch No.: 150001P040
- Expiration date of the lot/batch: April 23rd, 2016
- Stability under test conditions: Proven for 5h, the maximum time between preparation of dosing solution and dosing
- Storage condition of test material: At room temperature and protected from light

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: 10 to 14 weeks
- Weight at study initiation: 184 on average
- Fasting period before study: no
- Housing: individually in Macrolon plastic cages (MIII type)
- Diet (e.g. ad libitum): pelleted rodent diet ad lib. (SM R/M-Z from SSNIFF®)
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test substance. A correction was made for the purity/composition of the test substance. A correction factor of 1.28 was used. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 0.75, 2.5, 7.5%
- Amount of vehicle (if gavage): 4mL/kg b.w.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- No test substance was detected in the Group 1 formulations.
- The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
- The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%).
- Formulations at the entire range were stable when stored at room temperature protected from light for at least 5 hours (i.e. relative difference ≤ 10%).
- The long term storage samples were stable at ≤-70°C for 337 hours.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
Days 6 to 20 post-coitum, inclusive
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 100, 300mg/kg b.w.
Basis:
actual ingested
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on an OECD 422 study with dose levels up to 250 mg/kg bw/day, but without toxicity in females. The high dose level in the current study (300 mg/kg bw/day) is based on erosions of the forestomach seen in 1 out of 4 females dosed with 250 and 500 mg/kg bw/day after 14 days administration, respectively.

- Animal randomization: On the day of receipt, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the mean per subgroup. Females which were mated on the same day are classified in the same subgroup.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and at least once daily for clinical signs
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Days 1, 3, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Days 1-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Subjective appraisal, no quantitative measurements

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Reproductive organs, stomach, all further macroscopic anomalies

OTHER: Blood was collected directly prior to necropsy
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Placenta weight (for live fetuses): Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number and distribution of early resorptions: Yes
- Number and distribution of late resorptions: Yes
- Number and distribution of life fetuses: Yes
- Fetal weight: Yes
- Sex of each fetus: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) / number of corpora lutea * 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) / number of implantation sites * 100
Viable fetuses affected / litter (%) = (number of viable fetuses affected per litter) / number of viable fetuses per litter * 100
Historical control data:
Available for 5 studies performed in 2014/2015

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No mortality occured.
Salivation was observed in 12/22 females treated with 100mg/kg and all females treated with 300mg/kg. In addition, piloerection was seen in 1/22 and 3/22 animals treated at 100 and 300 mg/kg bw/day, respectively. Salivation was likely related to the taste or irritating properties of the test formulation applied and was not considered an adverse effect. In addition, piloerection was considered of no toxicological significance based on its low incidence within both dose groups.
There were no differences in body weight, body weight gain, or food consumption. Macroscopic examination revealed no substance related effect.
There was also no effect on the number of corpora lutea, implantation sites, or pre- or post-implantation loss in all dose groups. All females were pregnant with viable fetuses.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Mean litter sizes were 10.2, 11.5, 10.6 and 10.4 fetuses/litter for the vehicle control, 30, 100 and 300 mg/kg bw/day groups, respectively.
Mean sex ratios (males:females) were 51:49, 51:49, 57:43 and 42:58 for the vehicle control, 30, 100 and 300 mg/kg bw/day, respectively.
Mean combined (male and female) fetal body weights were 5.3, 5.2, 5.3 and 5.2 grams for the vehicle control, 30, 100 and 300 mg/kg bw/day, respectively.
Mean male/female placenta weights were 0.44/0.42, 0.45/0.43, 0.46/0.44 and 0.45/0.42 grams for the vehicle control, 30, 100 and 300 mg/kg bw/day, respectively.
The numbers of fetuses (litters) available for morphological examination were 224(22), 254(22), 234(22) and 228(22) in Groups 1, 2, 3, and 4, respectively.
There were no external developmental malformations or variations for any fetuses following treatment up to 300 mg/kg/day.
There were no treatment related effects on visceral morphology following treatment up to 300 mg/kg. Visceral malformations were noted in one fetus each of the low and high dose group. Visceral variations were observed for 8.6%, 6.2%, 6.7% and 5.1% of fetuses per litter in the control, 30, 100 and 300 mg/kg groups, respectively.
There were no treatment related effects on skeletal morphology following treatment up to 300 mg/kg. Skeletal malformations were noted in one fetus in each of the control, 30, 100 and 300 mg/kg groups. Skeletal variations were observed for 87.9%, 82.0%, 84.5% and 81.4% of fetuses per litter in the control, 30, 100 and 300 mg/kg groups, respectively.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion