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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:
05 Mar - 27 Nov 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).
Deviations:
no
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): Lanolin Fatty Acids (LANFA 2)
- Physical state: brown waxy solid
- Analytical purity: > 90%
- Purity test date: 18 Jul 2012
- Lot/batch No.: 0000678698
- Expiration date of the lot/batch: 18 Jul 2013
- Storage condition of test material: Room temperature in the dark, in the original container, light protected
- Stability of test item in vehicle: 8 days

Test animals

Species:
rat
Strain:
other: RccHanTM:WIST(SPF)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B. V., Horst, The Netherlands
- Age at study initiation: 11 weeks
- Weight at day 0 (post coitum): 196-246 g (females)
- Housing: single housing in Makrolon type-3 cages after the acclimatisation period
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 56/12) rodent maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The solutions were prepared daily. The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (15 - 25 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 20, 60, and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw/day
- Batch No.: 492194511, 103197718
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day, samples of the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Samples of about 1 g of each concentration were taken 5 hours and 8 days after commencement of dosing to confirm stability.
The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Harlan Laboratories Ltd. (Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analyzed using a GC method provided by the Sponsor.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 6 - 20 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
15 days, until day 21 of gestation
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were based on the results of a foregoing range finding study, in which animals were orally exposed to 100, 300, and 1000 mg/kg bw/day for 15 days. No adverse effects were observed up to the limit dose of 1000 mg/kg bw/day. Therefore, 100, 300, and 1000 mg/kg bw/day were selected as the dose levels for the main study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (viability, mortality), once daily (clinical signs)

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until day 21 post coitum

FOOD CONSUMPTION: Yes, recorded at 3-day intervals: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 21 post coitum
- Organs examined: Any female sacrificed or found dead during the study was subjected to macroscopic examination with emphasis on the uterus and its contents. Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
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
- Other: If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
The following statistical methods were used to analyze food consumption, body weights, reproduction and skeletal examination data:
- Means and standard deviations of various data were calculated and included in the report.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Indices:
Pre- and post-implantation losses
Historical control data:
Historical control data from several former embryofetal development toxicity studies were provided, giving information on skeletal findings, visceral examination, and reproductive data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
All females survived until the scheduled necropsy on day 21 post coitum.
No clinical symptoms or signs were observed at any dose level during the study that were considered to be related to the test item.
No test item-related changes were observed in the amount of food consumed during the study.
Absolute body weight and body weight gain were not affected by treatment with the test item. Corrected body weight gain (corrected for the gravid uterus weight) was not affected by treatment with the test item (+13.2%, +14.6%, +13.1%, 13.4% in order of ascending dose level).
The relevant reproduction data (post-implantation loss and number of fetuses per dam) were not affected by treatment with the test item.
No findings were observed at any dose level during macroscopical examination.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
No findings were observed during external examination of the fetuses in any group.
No test item-related effects on the sex ratio of the fetuses were noted in any group. The proportion of male fetuses was 52.9%, 46.4%, 53.2% and 49.1% in order of ascending dose level.
No test item-related effects on fetal body weights were noted. A marginal increase in group mean body weights of live fetuses calculated on an individual basis were seen for all test item treated groups when compared to controls, achieving statistical significance in groups 2 and 4. However, as the fetal weights were not statistically significant when calculated on a litter basis, were not dose-related, and since a toxicological effect is normally expected to cause a decrease in body weight, this was considered not to be a test item-related effect.

Visceral abnormalities and variations:
There were no findings seen during visceral examination that were considered to be related to maternal administration of the test item. Severe kidney pelvic dilatation was seen in one fetus in the group given 1000 mg/kg/day, and severely malpositioned testis was recorded for one fetus in the group given 100 mg/kg bw/day. However, as both instances were isolated findings, these were not considered to be test item-related.

Bone and cartilage abnormalities and variations:
No test item-related findings were observed.
Abnormalities were observed in 1 fetus from 1 litter at 100 mg/kg body weight/day (Skull bone supernumerary ossification site small or fused to the adjacent cervical vertebral archand; cervical and thoracic vertebral bodies/arches misshapen (partially split) / fused / absent). In addition abnormalities were observed in 2 fetuses from 2 litters at 1000 mg/kg bw/day (Skull bone supernumerary ossification site small or fused to the adjacent cervical vertebral arch; costal cartilage of cervical rib to thoracic rib fused). These abnormalities were all within historical background ranges and there was no dose-dependent pattern. Therefore the findings were considered to be incidental. Incidences of a long rib were observed in 3 fetuses from one litter. Since only 1 litter was affected, this was considered to be incidental.
Incidences of branched costal cartilage were seen in all treated groups, however were absent in controls. This was most prevalent in litters from the group administered 1000 mg/kg bw/day, with 5 fetuses (4%) in 4 litters (20%) effected. This incidence has been observed in this strain, and since no other bone effects have been observed this dose level, it is not considered to be a test item-related effect.
All other variations seen were considered to be incidental, with all values seen either within historical background ranges, non dosage related, at similar prevalence as seen in the controls or in a small number of fetuses spread between litters.

Ossification and supernumerary ribs:
No test item-related effects were observed. No statistically significance increases were observed in any of the findings at any dose level.

Additional cartilage variations:
There were no findings observed that were considered to be related to maternal administration of the test item. Although costal cartilage not reaching sternum was statistically significantly increased at 1000 mg/kg bw/day on a litter and on a fetus basis this incidence has been observed in this strain and is therefore not considered to be test item-related. All further findings were within the range of the historical control data or did not increase in a dose-dependent manner.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

 

Group 1 (control)

Group 2 (100 mg/kg bw/d)

Group 3 (300 mg/kg bw/d)

Group 4 (1000 mg/kg bw/d)

Number of dams

22

22

20

20

Corpora Lutea

Mean ± St. dev.

309

14±2

298

13.5±2.4

252

12.6±2.3

278

13.9±1.7

Pre-Implantation Loss

% of Corp. Lutea

Mean ± St. dev.

Number of dams affected

10

3.2

0.5±0.9

6

13

4.4

0.6±1.2

7

13

5.2

0.7±1.0

7

4

1.4

0.2±0.5

3

Implantation Sites

% of Corp. Lutea

Mean ± St. dev.

299

96.8

13.6±1.8

285

95.6

13.0±2.8

239

94.8

12.0±2.9

274

98.6

13.7±1.9

Post-Implantation Loss

% of Impl. Sites

Mean ± St. dev.

Number of dams affected

6

2

0.3±0.5

6

5

1.8

0.2±0.5

4

6

2.5

0.3±0.7

4

3

1.1

0.2±0.4

3

Implantation sites scars

0

0

0

0

Embryonic/Fetal Deaths total

6

5

6

3

Embryonic Resorptions

5

5

5

2

Fetal Resorptions

1

0

1

1

Total Fetuses

% of Impl. Sites

Mean ± St. dev.

293

98

13.3±1.8

280

98.2

12.7±2.9

233

97.5

11.7±3.0

271

98.9

13.6±1.9

Live Fetuses

293

280

233

271

Dead Fetuses

0

0

0

0

Abnormal Fetuses

0

0

0

0

Total Males

% of Fetuses

155

52.9

130

46.4

124

53.2

133

49.1

Total Females

% of Fetuses

138

47.1

150

53.6

109

46.8

138

50.9

Weight of Fetuses (Litter Basis)

 

 

 

 

Total Fetuses

N (Litters)

Mean ± St. dev.

 

22

4.8±0.3

 

22

5.0±0.3

 

20

4.9±0.4

 

20

5.0±0.2

Total Fetuses

N (Fetuses)

Mean ± St. dev.

 

293

4.8±0.4

 

280

4.9±0.4**

 

233

4.9±0.5

 

271

5.0±0.4**

*/**: Dunnett-Test based on pooled variance significant at level 5% (*) or 1% (**)

Applicant's summary and conclusion