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Toxicological information

Developmental toxicity / teratogenicity

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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented publication, the study was conducted under GLP conditions according to OECD guideline 414.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2008
Reference Type:
secondary source
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
dosing GD7-17
GLP compliance:
yes
Remarks:
According to the publication, all procedures were conducted in compliance with Good Laboratory Practice (GLP) regulations
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Linalool (Millennium Speciality Chemicals, Jacksonville, USA)
- Substance type: clear colourless liquid
- Analytical purity: 99.5 %
- Lot/batch No.: 5LM301
- Storage condition of test material: at room temperature and protected from light

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) IGS BR VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, USA)
- Weight at study initiation: females: 207 - 253 g
- Housing: individually in stainless steel, wire bottomed cages (except during the mating period, when each pair of male and female rat was housed in the male rat´s cage
- Diet: Certified rodent diet 5002 (PMI Nutrition International, St. Lous, USA), ad libitum
- Water: reverse-osmosis deionized water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 17.7 - 26.1 °C
- Humidity: 30 - 70 %
- Air changes: at least 10 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Dosing formulations were prepared weekly from bulk materials. Corn oil (lot number 015K0115; Sigma-Aldrich St. Lois USA) was used as vehicle and served as control.
Healthy, mated female rats were assigned to 4 dosage groups, using a computer-generated (weight-ordered) randomization procedure, based on body weights recorded on the day on which sperm was found in the vaginal smear or a copulatory plug was found in the vagina.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from each concentration of the dosing suspensions (first and last days of treatment) were analyzed for linalool content by Charles River Laboratories Preclinical Services (Worcester, USA).
Details on mating procedure:
During the mating period, one male and one female were housed in the male rat´s cage.
The presence of spermatozoa or a copulatory plug was designated as gestational day 0 (GD 0).
Duration of treatment / exposure:
The test material in corm oil vehicle was administered by gavage to 4 groups of pregnant rats from GD 7 to 17. The dosage volume was 10 ml/kg bw, adjusted daily according to the individual body weights recorded directly before gavage and administered approx. the same time each day.
Frequency of treatment:
once daily
Duration of test:
until GD 21
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
range-finding study: 0, 125, 250, 500 and 1000 mg/kg bw/d
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
main study: 0, 250, 500 and 1000 mg/kg bw/d
Basis:
nominal conc.
No. of animals per sex per dose:
25 female rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dosages for the main study were selected based on a range-finding study.

Examinations

Maternal examinations:
Animals were observed twice daily for viability and examined for clinical signs, abortions, and premature deliveries before dosage administration and approximately one hour later. Body weights were recorded prior to the start of the study and daily during the dosage and postdosage periods. Feed consumption was recorded on GDs 0, 7, 10, 12, 15, 18, and 21. On GD 21, all rats were euthanized by inhalation of carbon dioxide. Following caesarean sectioning, a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed.
Ovaries and uterine content:
Uteri of apparently nonpregnant rats were examined while pressed between glass plates to confirm the absence of implantation sites. Uteri from pregnant rats were excised and examined for number and distribution of implantations, live and dead fetuses, and early and late resorptions. The number of corpora lutea in each ovary was recorded.
Fetal examinations:
Fetuses were removed from the uterus, weighed, and examined for gender and gross external alterations. Live fetuses then were euthanized by an intraperitoneal injection of pentobarbital before undergoing further examination. Approximately half of the fetuses in each litter were fixed in Bouin’s solution and examined for soft tissue alterations, using a variation of Wilson’s sectioning technique . The remaining fetuses in each litter were eviscerated, cleared, stained with alizarin red S, and examined for skeletal alterations.
Statistics:
Data generated during the course of study were recorded either by hand or using the Argus Automated Data Collection and Management System and the Vivarium Temperature and Relative Humidity Monitoring System. All data were tabulated, summarized, and/or statistically analyzed using the above systems in conjunction with Microsoft Excel (Microsoft Office 97/2000XP), Quattro Pro 8, and/or The SAS System (version 6.12). Clinical observation and other proportion data were analyzed using the variance test for homogeneity of the binomial distribution. Continuous data were analyzed using Bartlett’s test of homogeneity of variances and the analysis of variance , when appropriate. Dunnett’s test was used to identify statistical significance of differences among individual groups. If the analysis of variance was not appropriate, the Kruskal-Wallis test or Dunn’s method of multiple comparisons was used to identify the statistical significance of differences among the individual groups. If there were greater than 75% ties, Fisher’s exact test was used to analyze the data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
No maternal mortality occurred during the study. There were no clinical signs of toxicity. All abnormal necropsy observations on GD 21 were considered unrelated to linalool because they occurred in single animals and were unrelated to dose.
No statistically significant differences occurred in mean body weights at necropsy or body weight gains at dosages as high as 1000 mg/kg/day. However, compared to vehicle control group values, mean body weight gains were reduced by 11% in the 1000 mg/kg/day dosage group during the dosing period, whereas a compensatory 7 % increase occurred during the postdosage period (GDs 18 to 21). Mean maternal relative and absolute feed consumption values paralleled the body weight gains, with a statistically significant reduction at some time points in the 1000 mg/kg/day dosage group.
Pregnancy occurred in 22 (88 %), 23 (92 %), 20 (80 %), and 22 (84 %) rats in the four respective dosage groups. One dam in the 250 mg/kg/day dosage group delivered early on the day of scheduled sacrifice; as a result, caesarean-sectioning observations on GD 21 were based on 22, 22, 20, and 22 deliveries. The early delivery was considered unrelated to linalool because this incident was not dose dependent, and no adverse clinical signs or abnormal feed consumption or body weight values were observed prior to delivery.
There were no gross lesions at necropsy, and the litter for this dam consisted of 12 live-delivered pups and one early in utero resorption.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
All pups appeared normal and no gross external, soft tissue, or skeletal alterations were noted.
No caesarean-sectioning or litter parameters were affected by dosages of linalool as high as 1000 mg/kg/day. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, percent resorbed conceptuses, percent live fetuses, and fetal body weights were comparable among the four dosage groups and did not significantly differ. There were four dead fetuses in the 0 (vehicle) mg/kg/day dosage group; no other dead fetuses were observed. No dam had a litter that consisted of all resorbed conceptuses. All placentae appeared normal.
Fetal evaluations were based on the 277, 293, 254, and 271 live GD 21 caesarean-delivered fetuses in 22, 22, 20, and 22 litters in the 0, 250, 500, and 1000 mg/kg/day dosage groups, respectively. Each of these fetuses was examined for gross external alterations, whereas 135, 141, 122, and 127 were examined for soft tissue alterations, 146, 152, 132, and 144 were examined for skeletal alterations, and 143, 152, 132, and 144 were examined for ossification site averages ( Fetal alterations: malformations or variations).
Gross external alterations included: unilateral cleft palate and whole-body edema in one fetus at 1000 mg/kg/day and a shortened trunk in a fetus at 250 mg/kg/day. Soft tissue changes included only one malformation, microphthalmia (one eye) in one control fetus, which also had a slight dilation of the renal pelvis (a variation that also occurred in another control fetus and in one fetus at 500 mg/kg/day). Umbilical artery variations were present in 0, 2, 2, and 1 fetus from the 0, 250, 500, and 1000 mg/kg dosage groups, respectively.
Skeletal malformations included a cleft palate, which was incompletely ossified, as were the right premaxilla and maxilla, in the edematous previously mentioned fetus at 1000 mg/kg/day. This fetus also had multiple skeletal alterations, including two incompletely ossified sternebral centra; irregular or fused arches in multiple vertebrae; missing thoracic vertebrae; unattached or fused, close-set ribs; and irregular or missing lumbar vertebrae. Fused arches in one or more thoracic vertebrae and a lack of ossification in two sternal centra and in the centrum of the third thoracic vertebra were also observed in one fetus at 500 mg/kg/day, whereas multiple rib and vertebral alterations were noted in the fetus with the shortened body at 250 mg/kg/day.
Several minor skeletal variations occurred in a few other control or treated fetuses, but the incidences were not dosage or treatment related.

The noted gross external, soft tissue, or skeletal fetal alterations (malformations or variations) were not considered to be caused by the test material because these changes were not dosage dependent, did not significantly differ from vehicle control group values, and the incidences were within the ranges observed historically at the Testing Facility. In 101 studies (1685 litters; 24,434 fetuses) conducted at the Testing Facility during a 2-year period just prior to when the study was conducted, cleft palate occurred in a total of three control group fetuses, one in each of three different studies (litter incidence = 0.18%; range per study = 0 to 1 [0% to 4.3%]; fetal incidence = 0.01%; range per study = 0 to 1 [0% to 0.3%]). Whole body edema occurred in a total of three control group fetuses, one in each of three different studies (litter incidence = 0.18%; range per study = 0 to 1 [0% to 4.3%]; fetal incidence = 0.01%; range per study = 0 to 1 [0% to 0.3%]). All ossification averages were comparable to vehicle control group values and did not significantly differ among the groups.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Results of concentration analyses from the start and end of study revealed that all prepared formulations were within the acceptable limits of ± 15 %. All homogeneity results were within the acceptable range of ≤ 2 % relative standard deviation (RSD).

In the range-finding study, an 8 % decrease in feed consumption at 1000 mg/kg/day during the dosage period was the only adverse effect noted in the dams. There were no untoward changes noted in caesarean sectioning or litter parameters, and no fetal external alterations were found at any dosage level. Based on these data, dosages of 0, 250, 500, and 1000 mg/kg/day of the test material were selected for the definitive developmental toxicity study in rats.

Table 1: Effect on oral administration of the test material on maternal absolute feed consumption and maternal body weight gains

 

Maternal absolute feed consumption (g/d; mean ± SD)

 

dosage group (mg kg bw/d)

 

0 (control)

250

500

1000

GD 0-7

22.4 ± 2.1

22.6 ± 2.5

22.4 ± 2.6

22.5 ± 1.8

GD 7-10

15.8 ± 2.7

17.2 ± 2.7

16.4 ± 3.5

13.4 ± 1.8 **

GD 10-12

17.8 ± 2.8

18.4 ± 2.7

18.3 ± 3.6

17.6 ± 2.4

GD 12-15

18.6 ± 3.1

18.3 ± 2.5

18.2 ± 2.8

17.9 ± 2.1

GD 15-18

20.5 ± 3.6

19.8 ± 3.1

19.4 ± 3.4

18.7 ± 2.6

GD 18-21

23.4 ± 3.5

23.5 ± 2.9

22.9 ± 2.6

25.4 ± 1.8 *

 

Maternal body weight gains (g, mean ± SD)

 

dosage group (mg kg bw/d)

 

0 (control)

250

500

1000

GD 0-7

40.8 ± 6.3

42.4 ± 10.1

43.0 ± 9.5

38.9 ± 8.9

GD 7-10

9.0 ± 7.6

10.3 ± 6.8

11.2 ± 8.6

6.4 ± 7.3

GD 10-12

11.9 ± 5.3

12.8 ± 5.1

12.1 ± 4.5

12.9 ± 6.2

GD 12-15

17.2 ± 8.8

17.0 ± 7.6

15.9 ± 6.5

18.5 ± 7.3

GD 15-18

40.5 ± 11.4

36.7 ± 9.1

36.9 ± 10.4

31.9 ± 10.4

GD 18-21

36.2 ± 11.8

35.4 ± 21.0

36.4 ± 7.9

39.0 ± 11.9

∗ = significantly different from the vehicle control group value (p ≤ .05)

∗∗ = significantly different from the vehicle control group value (p ≤ .01)

Table 2: Effect of maternal administration of the test material on caesarian-section and litter data

 

dosage group (mg kg bw/d)

 

0 (control)

250

500

1000

rats tested

25

25

25

25

rats pregnant (%)

22 (88.0)

23 (92.0)

20 (80.0)

22 (88.0)

rats caesarian sectioned on GD 21

22

22

20

22

Corpora lutea (mean ± SD)

14.0 ± 3.4

15.5 ± 2.8

14.4 ± 3.5

14.3 ± 3.5

Implantations (mean ± SD)

13.1 ± 3.8

14.3 ± 2.3

13.4 ± 2.9

12.9 ± 4.1

Resorptions (mean ± SD)

0.4 ± 0.6

1.0 ± 1.7

0.6 ± 0.7

0.5 ± 0.8

Litter size (mean ± SD)

12.6 ± 4.2

13.3 ± 2.4

12.7 ± 3.0

12.3 ± 4.0

Fetal body weight/litter (mean ± SD)

5.1 ± 0.7

4.8 ± 0.5

4.9 ± 0.5

4.9 ± 0.5

live fetuses

277

293

254

271

dead fetuses

4

0

0

0

Table 3: Fetal alterations observed (gross external, soft tissue, or skeletal variations or malformations) following oral administration of the test material to pregnant rats

 

dosage group (mg kg bw/d)

 

0 (control)

250

500

1000

litters evaluated

22

22

20

22

fetuses evaluated

281

293

254

271

litters with fetuses with any observed alterations (%)

4 (18.2)

6 (27.3)

7 (35.0)

4 (18.2)

fetuses with any observed alterations

5 (1.8)

6 (2.0)

8 (3.1)

4 (1.5)

% fetuses/litter with any alteration (mean ± SD)

1.6 ± 3.6

2.1 ± 3.7

3.0 ± 4.7

1.5 ± 3.4

Applicant's summary and conclusion