Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
other: Official Journal of the European Communities, 87/302/EEC, Part B: Methods for the Determination of Toxicity.
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
other: MAFF Japan, 119 Nousan Number 4200, Agricultural Chemicals Laws and Regulations, Japan (II) (1985).
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.7%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 67 days
- Weight at study initiation: Range between 227.0-282.0 grams
- Fasting period before study: None
- Housing: Individually in stainless steel, wire-mesh cages suspended above cage boards. Cage racks were not relocated within the animal room.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 30%-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): Artificial (fluorescent light) on an approximate 12-hour light/dark cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations of the test substance in the vehicle were prepared at least once each week; formulations at the concentrations selected for the current study were previously demonstrated to be stable for 24 hours at room temperature and for 7 days in the refrigerator. The test substance was suspended in 0.5% aqueous methylcellulose. Dosing formulations were stored until used under conditions defined by the existing formulation stability data cited above (24 hours at room temperature, 7 days refrigerated).
VEHICLE The vehicle was 0.5% (w/v) aqueous methylcellulose. There were no known contaminants in the vehicle that would be expected to have had any adverse impact on the integrity of the study. The vehicle was assumed to be stable under the conditions of the study. The vehicle was stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analysed once near the beginning of the study and again near the end of the study. Analyses addressed uniformity of mixing and concentration. Samples were analysed shortly after preparation. Method of Analysis: gas chromatography (GC) with flame ionisation detector (FID).

Data from the analysis of the formulation samples indicated that the test substance was uniformly mixed in the vehicle at the targeted concentrations and was stable in the vehicle under the conditions of the study. Test substance was not found in the 0 mg/mL sample (control).
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
gestation days 6-20
Frequency of treatment:
Daily
Duration of test:
Through gestation day 21
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 25, 125, or 250 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
22 time-mated rats/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses selected for the current study were based on results of repeated-dose studies with the test substance. In a subchronic study, animals were dosed at the same dose levels selected for the current study using the same vehicle and dose volume. Test substance-related mortality occurred at 250 mg/kg/day; however, the earliest death did not occur until after 20 consecutive doses. In addition, for animals that died early, the animals appeared to tolerate dosing with minimal impact on body weight, food consumption, and clinical observations until just a few days before death. In a one-generation reproduction study similar toxicity was reported. The data indicate that 250 mg/kg/day may increase maternal mortality at the very end of gestation suggesting that these deaths might be related to difficulty with parturition. Since mortality was observed at 250 mg/kg/day in these previous studies, it was not deemed necessary to exceed a high dose level of 250 mg/kg/day. However, since the current study dosing period extends 15 consecutive days in length and maternal animals are euthanised prior to parturition, the high dose level of 250 mg/kg/day was expected to be tolerated. The lower doses were selected to explore the possible dose response relationship of any test substance-related findings as well as to establish a no-observed-effect level (NOEL).

- Rationale for animal assignment: Before dosing began, animals were randomly assigned to control or experimental groups using a computerised randomisation procedure to produce a homogeneous distribution of body weights across groups within each breeding lot.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily on GD 6-20 (during weighing and at least two hours post-dosing); Once on GD 21

BODY WEIGHT: Yes
- Time schedule for examinations: Daily on GD 6-21

FOOD CONSUMPTION: Yes
- Time schedule for examinations: GD 6, 8, 10, 12, 14, 16, 18, 20, and 21

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: A gross external and visceral examination was performed immediately after euthanasia.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [all per litter ]
- Head examinations: Yes: [ half per litter ]
Statistics:
See Table 1 below.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There was no test substance-related mortality at any level tested; all animals on study survived until scheduled euthanasia.
At 250 mg/kg/day, there was a slight increase in the incidences of stained and/or wet fur. At 125 mg/kg/day and lower, there were no test substance-related clinical observations; occasional instances of hair loss were unremarkable and considered unrelated to dose.
There were test substance-related reductions in maternal body weight parameters at 125 and 250 mg/kg/day. At 250 mg/kg/day, mean body weights were significantly lower than controls beginning on GD 7 and persisting until the end of gestation. Mean absolute final body weight and final body weight adjusted for the products of conception were 10 and 11% lower, respectively than the corresponding control group values. These reductions were the result of a cumulative weight gain that was lower than the control group gain by 24 or 50% using either the absolute or adjusted final weight to calculate the gain. At 125 mg/kg/day, mean body weights were slightly and/or significantly lower throughout gestation and resulted in a mean final body weight (absolute or adjusted) that was 4% lower than the corresponding control mean. This reduction was the result of lower weight gain; cumulative weight gain was 9 or 24% lower than controls using either the absolute or adjusted final weight to calculate the gain. At 5 and 25 mg/kg/day, maternal body weight and weight gain data were comparable to control group data.
There were test substance-related reductions in maternal food consumption at 125 and 250 mg/kg/day. At 125 and 250 mg/kg/day, mean food consumption was lower or significantly lower than controls for the entire dosing period resulting in a cumulative mean food consumption (GD 6 to 21) that was 8 and 12% lower than controls, respectively. There were no effects on maternal food consumption at 5 or 25 mg/kg/day; data from these groups were comparable to control group data.
There were no test substance-related maternal gross postmortem observations at any level tested. The observations that were recorded were unremarkable and occurred in single animals in any affected group.

Effect levels (maternal animals)

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

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
There were no test substance-related fetal malformations observed at any dose level tested.
At 125 and 250 mg/kg/day, the incidences of selected skeletal alterations were increased or significantly increased; the specific findings were incomplete ossification of skull bones and wavy and/or thickened ribs. At 250 mg/kg/day, the incidence of delayed pelvic bone ossification was also increased. See Table 2.
There were no test substance-related fetal alterations at 5 or 25 mg/kg/day. The fetal alterations that were observed at these levels were unremarkable, not dose-related, and occurred with low frequency.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 2: Skeletal Findings

 

Observations+

Dose (mg/kg bw/day)

0

5

25

125

250

#Fetuses(litters) examined (Head effects)

 144(22)

144(22)

142(22) 

142(22)

138(22)

Skull frontal, incomplete ossification

   

 

   

  1  (0.7 )

   

Skull Zygomatic, Incomplete ossification

 

 

 

 3 (2.1)

3 (2.2)

Skull Interparietal, Incomplete ossification

 

1 (0.7)

2 (1.4)

10 (7.0)

13 (9.4)

Skull, Supraoccipital, Incomplete ossification

 

 

 

11 (7.7)

30 (21.7)

Skull, Parietal, Incomplete ossification

1 (0.7)

 

 

7 (4.9)

17 (12.3)

#Fetuses(litters) examined (Body effects)

 278(22)

277(22)

270(22) 

275(22)

267(22)

Wavy ribs

 

 

 

2 (0.7)

2 (0.7)

Thickened ribs

 

 

 

 7 (2.5)

 3 (1.1)

Pubis, Incomplete ossification

 

1 (0.4)

 

1 (0.4)

5 (1.9)

Ischium, Incomplete ossification

 

1 (0.4)

 

1 (0.4)

3 (1.1)

Applicant's summary and conclusion

Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
There was no evidence of either maternal or developmental toxicity at 5 or 25 mg/kg/day. Therefore, under the conditions of the current study, the no-observed-adverse-effect level (NOAEL) for both the dam and the fetus was 25 mg/kg/day.
Executive summary:

Groups of 22 time-mated rats were administered formulations of the test substance on days 6 through 20 of gestation at dose levels of 0, 5, 25, 125, or 250 mg/kg/day. Doses were formulated in 0.5% aqueous methylcellulose and administered at a dose volume of 5 mL/kg. Dose formulations were determined to be homogeneous, stable under conditions of use, and at targeted concentrations. During the in-life period, data collection included body weights, food consumption, and clinical observations. On day 21 of gestation, dams were euthanised and examined grossly. Gravid uterine weight was recorded and the uterine contents described and removed. Corpora lutea, implantation sites, and resorptions were counted for each litter. Live fetuses were individually identified and weighed. Fetuses were examined for external, visceral, and skeletal alterations.

Maternal and developmental toxicity were observed at 125 and 250 mg/kg/day. At 250 mg/kg/day, maternal toxicity was evident as reduced body weight and food consumption. The cumulative mean weight gain (gestations days 6 to 21) was 24% lower than the control group mean. Cumulative weight change calculated using maternal body weight adjusted for the weight of the products of conception was 50% lower than controls. Mean food consumption for the same interval was 12% lower than controls. At 250 mg/kg/day, there was also a slightly increased incidence of stained and/or wet fur. Similar evidence of maternal toxicity was produced at 125 mg/kg/day. Cumulative weight gain was 9 and 21% lower than controls, using either the absolute weight gain or the gain calculated using the adjusted final body weight, respectively. Mean food consumption for the same interval was 8% lower than controls. Evidence of developmental toxicity was limited to test substance-related increases in skeletal variations (ossification delays in the skull and rib alterations) at 125 and 250 mg/kg/day. At 250 mg/kg/day, there was a slight elevation in the incidence of pelvic bone ossification. There was no evidence of either maternal or developmental toxicity at 5 or 25 mg/kg/day. Therefore, under the conditions of the current study, the no-observed-adverse-effect level (NOAEL) for both the dam and the fetus was 25 mg/kg/day.