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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: The study was conducted according to generally valid and/or internationally accepted testing guidelines.
Justification for type of information:
No reproductive toxicity studies have been conducted on Dihydromyrcenol. For read-across purposes, data on a similar substance is provided
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid and/or internationally accepted testing guidelines.
Justification for type of information:
Read-across from Supporting substance (structural analogue or surrogate)
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
other: US FDA; This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C and D of the reproductive process
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Cr1:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at arrival: 62 days (approximate)
- Weight at study initiation: 218 to 244 g
- Fasting period before study: None
- Housing: Individually in stainless steel, wire-bottomed cages (except during cohabitation)
- Diet (ad libitum): Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO)
- Water (ad libitum): local water processed by reverse osmosis. Chlorine added as bacteriostat
- Acclimation period: Yes, but duration not stated


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64 to 79 (targeted)
- Humidity (%): 30 to 70 (targeted)
- Air changes (per hr): 10 (minimum)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test article were prepared weekly at the testing facility. Prepared formulations were stored at room temperature, protected from light.

VEHICLE
- Concentration in vehicle: The dosage preparations are shown in Table 1.
- Amount of vehicle (if gavage): Constant dosage volume of 10 ml/kg
Details on analytical verification of doses or concentrations:
Samples were analyzed according to the method described in Charles River Laboratories Preclinical Services. Results of concentration and homogeneity analyses were within +/- 15% of calculated concentrations and
Details on mating procedure:
After a suitable acclimation, 130 virgin female rats were placed into cohabitation with 130 breeder male rats, one male rat per female rat. The cohabitation period was a maximum of five days. Gestation day 0 was considered the first day when spermatozoa were detected in a vaginal smear or a copulatory plug was observed. Pregnant rats were assigned to individual housing.
Duration of treatment / exposure:
Rats were treated on gestation days 7 through 17. Dosages were adjusted daily based on the individual body weights recorded prior to dosage administration.
Frequency of treatment:
Once daily
Duration of test:
Until gestation day 21.
No. of animals per sex per dose:
25 female/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of a range-finding study with the same test article at a dosage level of 1000 mg/kg/day. This was selected as the highest dose in the current study.
- Rationale for route of administration: Oral (gavage) was selected for use as comparison with the dietary route; the exact dosage can be accurately determined.
Maternal examinations:
Rats were observed for viability at least twice daily each day of the study and weekly for clinical observations and general appearance during the acclimation period and on DG 0. Rats were examined for clinical observations, abortions, premature deliveries and deaths before and approximately 3 hours after dosing, and once daily during the post-dosage period.

Body weights were recorded weekly during the acclimation period, on DG 0 and daily during the dosage and post-dosage periods. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18 and 21.

All female rats were sacrificed by carbon dioxide asphyxiation on DG 21. Gross lessions were retained in neutral-buffered 10% formalin for possible future evaluation. Unless specifically cited, all other tissues were discarded.
Ovaries and uterine content:
At necropsy, females were Caesarian-sectioned and gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Uteri of apparently nonpregnant rats were examined while pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries of apparently nonpregnant rats were retained in neutral buffered 10% formalin and discarded when directed by the Study Director.

The number and distribution of corpora lutea were recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantation sites, early and late resorptions and live and dead fetuses. Early resorption was defined as one in which organogenesis was not grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Dead fetuses and late resorptions were differentiated by the degree of autolysis present. Placentae were examined for size, color and shape.
Fetal examinations:
Each fetus was removed from the uterus and individually stored and identified. Each was subsequently weighed and examined for gender and gross external alterations. Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. Approximately 1/2 of the fetuses in each litter were examined for soft tissue alterations using a variation of the adaptation of Wilson's sectioning technique. These fetuses were fixed in Bouin's solution and sections retained in alcohol. The remaining fetuses were evicerated, cleared, stained with alizarin red S and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal preparations were retained in glycerin with thymol added.
Statistics:
Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., body weights, body weight changes, feed consumption values, and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyze the data. Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Indices:
Maternal absolute and relative feed consumption.

Litter observations included fetal weights (male and female) and counts of corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, and resorbed conceptuses.
Historical control data:
Historical control data was available from this laboratory for the period from January 2005 to January 2007.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All female rats survived until scheduled sacrifice and there were no clinical observations that were considered related to test article administration. There were no gross lesions reported.

Weight losses were recorded on the first 2 days of dosing at the 1000 mg/kg bwt/day dose level, resulting in 5% reductions over control values. Overall weight gain was 5% less than the vehicle control group for the entire dosage period (days 7 to 18). Reductions in maternal weight gains were not statistically significant (Table 2).

Reductions in body weight gains were associated with significant reductions in absolute and relative feed consumption values at the 1000 mg/kg bwt/day dose level (Table 3). These occurred on gestation days 7 to 10, 10 to 12 and for the entire dosage period as compared with vehicle controls.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The 1000 mg/kg bwt/day dose level represented an apparent threshold for developmental toxicity as evidenced by an approximately 3% reduction in fetal body and an associated retardation in ossification of the metatarsal bones. These variations were within historical ranges and were not considered of toxicological significance.

Reflecting the slight maternal toxicity noted, there were increases in supernumerary thoracic ribs, with associated increases and decreases in thoracic and lumbar vertebrae, respectively. These variations are reversible and are often observed at maternally toxic doses (Table 4).
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: Developmental toxicity - Minimal reductions in foetal weights and small increase in reversible variations in skeletal
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 2: MATERNAL BODY WEIGHT GAINS - SUMMARY

DOSE GROUP

I

II

III

IV

DOSAGE (mg/kg bwt/day)

0 (control)

250

500

1000

RATS TESTED

N

25

25

25

25

PREGNANT

N

22

24

24

25

MATERNAL BODY

WEIGHT CHANGE (G)

DAYS 0 - 7

MEAN±S.D.

+42.5 ± 7.2

+42.0 ± 8.2

+40.7 ± 10.6

+39.8 ± 7.5

DAYS 7 - 8

MEAN±S.D.

-2.6 ± 4.6

-0.4 ± 5.2

-1.2 ± 5.5

-4.1 ± 6.8

DAYS 8 - 9

MEAN±S.D.

+1.4 ± 4.3

+2.0 ± 5.6

+1.5 ± 4.0

-1.1 ± 6.2

DAYS 9 - 10

MEAN±S.D.

+3.2 ± 4.6

+4.0 ± 4.8

+6.1 ± 4.2

+5.4 ± 5.7

DAYS 7 - 10

MEAN±S.D.

+2.0 ± 6.5

+5.5 ± 7.0

+6.4 ± 8.6

+0.1 ± 9.4

DAYS 10 - 12

MEAN±S.D.

+12.0 ± 5.3

+14.8 ± 5.4

+12.9 ± 5.2

+11.8 ± 9.6

DAYS 12 - 15

MEAN±S.D.

+17.7 ± 8.5

+17.1 ± 9.5

+19.8 ± 5.8

+18.4 ± 7.3

DAYS 15 - 18

MEAN±S.D.

+41.2 ± 9.6

+39.4 ± 8.8

+41.2 ± 7.3

+39.0 ± 7.7

DAYS 7 - 18

MEAN±S.D.

+73.0 ± 17.0

+76.8 ± 15.2

+80.2 ± 14.5

+69.3 ± 15.1

DAYS 18 - 21

MEAN±S.D.

+59.4 ± 11.4

+54.2 ± 8.7

+61.0 ± 10.8

+58.1 ± 12.8

DAYS 7 - 21

MEAN±S.D.

+132.4 ± 22.6

+130.9 ± 16.7

+141.2 ± 20.5

+127.4 ± 20.5

DAYS 0 - 21

MEAN±S.D.

+174.9 ± 25.6

+173.0 ± 21.0

+181.9 ± 27.2

+167.2 ± 23.6

Table 3: MATERNAL RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - SUMMARY

DOSAGE GROUP

I

II

III

IV

DOSAGE (MG/KG/DAY)a

0 (VEHICLE)

250

500

1000

RATS TESTED

N

25

25

25

25

PREGNANT

N

22

24

24

25

MATERNAL FEED

CONSUMPTION (G/KG/DAY)

DAYS 0 - 7

MEAN±S.D.

96.2 ± 6.8

94.2 ± 6.9

93.5 ± 10.6

92.5 ± 6.5

[ 23]b

[ 23]b

DAYS 7 - 10

MEAN±S.D.

58.6 ± 10.2

60.8 ± 9.7

60.4 ± 12.7

46.2 ± 11.3**

[ 23]b

DAYS 10 - 12

MEAN±S.D.

61.4 ± 9.6

65.4 ± 8.8

60.0 ± 7.9

54.2 ± 15.5*

DAYS 12 - 15

MEAN±S.D.

59.8 ± 7.2

63.5 ± 9.7

59.6 ± 7.1

54.9 ± 9.3

[ 22]b

[ 23]b

DAYS 15 - 18

MEAN±S.D.

63.8 ± 14.0

68.7 ± 7.6

64.2 ± 10.0

60.8 ± 8.9

[ 21]b

DAYS 7 - 18

MEAN±S.D.

61.0 ± 5.6

64.5 ± 6.0

61.4 ± 7.3

54.1 ± 6.1**

[ 21]b

DAYS 18 - 21

MEAN±S.D.

73.1 ± 8.1

71.8 ± 8.7

74.6 ± 7.1

76.2 ± 8.1

DAYS 7 - 21

MEAN±S.D.

63.7 ± 4.8

66.4 ± 5.4

64.7 ± 6.0

59.6 ± 5.3*

DAYS 0 - 21

MEAN±S.D.

69.5 ± 4.0

70.5 ± 4.6

69.0 ± 5.3

66.0 ± 3.8*

[ ] = NUMBER OF VALUES AVERAGED

a. Dosage occurred on days 7 through 17 of gestation.

b. Excludes values that could not be calculated or appeared incorrectly recorded, as well as those associated with spillage.

* Significantly different from the vehicle control group value (p≤0.05). ** Significantly different from the vehicle control group value (p≤0.01).

Table 4: Dosage-Dependent Statistically Significant Fetal Observations (Average Ossification Sites per Fetus per Litter)

Observation

Dosage Group (DGs 7 through 17)

mg/kg/day

0 (Vehicle)

250

500

1000

Historical Controla

Litters

22

24

24

25

982

Fetuses

155

164

183

184

7305

Rats tested, n

25

25

25

25

Rats pregnant, n (%)

22 (88)

24 (96)

24 (96)

25 (100)

Corpora lutea, mean ± SD

14.6 ± 2.4

14.9 ± 2.8

16.3 ± 2.4

15.8 ± 2.3

Implantations, mean ± SD

14.0 ± 2.1

13.8 ± 2.8

15.2 ± 1.7

14.8 ± 1.8

Resorptions, mean ± SD

0.6 ± 0.7

0.8 ± 0.9

0.6 ± 0.6

0.5 ± 1.2

Litter size, mean ± SD

13.4 ± 2.2

13.0 ± 2.4

14.6 ± 2.0

14.3 ± 2.4

Fetal body weight/litter, mean ± SD

5.50 ± 0.28

5.54 ± 0.30

5.55 ± 0.31

5.33 ± 0.33

Male fetuses

5.63 ± 0.28

5.73 ± 0.31

5.68 ± 0.28

5.45 ± 0.36

Female fetuses

5.38 ± 0.29

5.37 ± 0.28

5.42 ± 0.35

5.18 ± 0.32*

Live fetuses/group

295

311

350

358

Dead fetuses/group

0

0

0

0

Ribs (pairs)b,c

13.06 ± 0.11

13.06 ± 0.10

13.09 ± 0.13

13.19 ± 0.23d

13.06 (13.02-13.12)

Thoracic Vertebraeb,c

13.08 ± 0.15

13.08 ± 0.16

13.12 ± 0.17

13.24 ± 0.28*,d

13.08 (13.02-13.16)

Lumbar Vertebraeb,c

5.92 ± 0.15

5.91 ± 0.16

5.87 ± 0.17

5.75 ± 0.27**,d

5.92 (5.83-5.97)

Metatarsalsb,c

4.95 ± 0.12

4.87 ± 0.21

4.91 ± 0.12

4.78 ± 0.27**,d

4.83 (4.68-4.95)

a. Studies (N = 44) conducted at the Testing Facility between January 2005 and January 2007 in rats of the same strain.

b. Means and Standard Deviations

c. Mean and range of means per study

d. Bold = effect of test article because values were significant and/or exceeded historical ranges. DGs = Days of Gestation

* = Significantly different from the vehicle control group value (p≤0.05). ** = Significantly different from the vehicle control group value (p≤0.01).

Testing facility historical control data for mean fetal body weight ranges (82 studies): combined male/female, 4.94 to 5.93 g; male fetuses, 5.10 to 5.98 g; female fetuses, 4.80 to 5.78 g.

Conclusions:
On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) was 1000 mg/kg bwt/day. At this dose level, maternal body weight gains were reduced (not significant) and feed consumption was significantly reduced. The 500 mg/kg bwt/day dose level was assigned as the NOEL for maternal effects.

The developmental NOAEL was also 1000 mg/kg bwt/day. This was based on minimal reductions in female fetal weights and small increases in reversible variations in skeletal ossification. The 500 mg/kg bwt/day dose level was assigned as the NOEL for developmental effects.
Executive summary:

Dimyrcetol was administered in corn oil by gavage to groups of pregnant Sprague-Dawley rats at dosages of 0, 250, 500 or 1000 mg/kg bwt/day on gestational days 7 to 17. Viability, clinical signs, body weights and feed consumption were observed or measured for all animals. Animals were sacrificed on gestational day 21 and Caesarean sectioning performed. Fetuses were weighed and examined for sex, gross changes, and soft tissue or skeletal alterations. No clinical signs attributable to dimyrcetol administration were observed. At the 1000 mg/kg bwt/day dose level, mean maternal body weight gains were reduced 5% versus controls. Absolute and relative feed consumption were also reduced at this dose level. The minimal maternal effects noted were associated with minimal (approx. 3%) reductions in fetal body weights, reversible variations in ossification and increases in supernumerary thoracic ribs with associated increases or decreases in thoracic and lumbar vertebrae, respectively. Findings of delayed skeletal ossification are generally considered readily reversible (Carney and Kimmel, 2007 "Interpretation of skeletal variations for human risk assessment: Delayed ossification and wavy ribs", Birth Defects Research 80:473 -496). The minimal developmental delays (reduced pup weights, delayed ossificaiton, changes in metatarsals) are all expected to be readily reversible based on studies that have investigated the progression and outcome of similar effects.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: US FDA; This study was designed to evaluate ICH Harmonised Tripartite Guideline stages C and D of the reproductive process
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: identified as Dihydromyrcenol
- Composition of test material, percentage of components: 2,6-dimethyl-7-octen-2-ol formate (54.8%) and 2,6-dimethyl-7-octen-2-ol (44.2%)
- Lot/batch No.: 4103311
- Stability under test conditions: Under the specified storage conditions, stability for 10 days was confirmed.
- Storage condition of test material: Stored at room temperature protected from light.

Test animals

Species:
rat
Strain:
other: Cr1:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at arrival: 62 days (approximate)
- Weight at study initiation: 218 to 244 g
- Fasting period before study: None
- Housing: Individually in stainless steel, wire-bottomed cages (except during cohabitation)
- Diet (ad libitum): Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO)
- Water (ad libitum): local water processed by reverse osmosis. Chlorine added as bacteriostat
- Acclimation period: Yes, but duration not stated


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64 to 79 (targeted)
- Humidity (%): 30 to 70 (targeted)
- Air changes (per hr): 10 (minimum)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test article were prepared weekly at the testing facility. Prepared formulations were stored at room temperature, protected from light.

VEHICLE
- Concentration in vehicle: The dosage preparations are shown in Table 1.
- Amount of vehicle (if gavage): Constant dosage volume of 10 ml/kg
Details on analytical verification of doses or concentrations:
Samples were analyzed according to the method described in Charles River Laboratories Preclinical Services. Results of concentration and homogeneity analyses were within +/- 15% of calculated concentrations and
Details on mating procedure:
After a suitable acclimation, 130 virgin female rats were placed into cohabitation with 130 breeder male rats, one male rat per female rat. The cohabitation period was a maximum of five days. Gestation day 0 was considered the first day when spermatozoa were detected in a vaginal smear or a copulatory plug was observed. Pregnant rats were assigned to individual housing.
Duration of treatment / exposure:
Rats were treated on gestation days 7 through 17. Dosages were adjusted daily based on the individual body weights recorded prior to dosage administration.
Frequency of treatment:
Once daily
Duration of test:
Until gestation day 21.
No. of animals per sex per dose:
25 female/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of a range-finding study with the same test article at a dosage level of 1000 mg/kg/day. This was selected as the highest dose in the current study.
- Rationale for route of administration: Oral (gavage) was selected for use as comparison with the dietary route; the exact dosage can be accurately determined.

Examinations

Maternal examinations:
Rats were observed for viability at least twice daily each day of the study and weekly for clinical observations and general appearance during the acclimation period and on DG 0. Rats were examined for clinical observations, abortions, premature deliveries and deaths before and approximately 3 hours after dosing, and once daily during the post-dosage period.

Body weights were recorded weekly during the acclimation period, on DG 0 and daily during the dosage and post-dosage periods. Feed consumption values were recorded on DGs 0, 7, 10, 12, 15, 18 and 21.

All female rats were sacrificed by carbon dioxide asphyxiation on DG 21. Gross lessions were retained in neutral-buffered 10% formalin for possible future evaluation. Unless specifically cited, all other tissues were discarded.
Ovaries and uterine content:
At necropsy, females were Caesarian-sectioned and gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Uteri of apparently nonpregnant rats were examined while pressed between glass plates to confirm the absence of implantation sites. Uteri and ovaries of apparently nonpregnant rats were retained in neutral buffered 10% formalin and discarded when directed by the Study Director.

The number and distribution of corpora lutea were recorded. The uterus of each rat was excised and examined for pregnancy, number and distribution of implantation sites, early and late resorptions and live and dead fetuses. Early resorption was defined as one in which organogenesis was not grossly evident. A live fetus was defined as a term fetus that responded to stimuli. Dead fetuses and late resorptions were differentiated by the degree of autolysis present. Placentae were examined for size, color and shape.
Fetal examinations:
Each fetus was removed from the uterus and individually stored and identified. Each was subsequently weighed and examined for gender and gross external alterations. Live fetuses were sacrificed by an intraperitoneal injection of sodium pentobarbital. Approximately 1/2 of the fetuses in each litter were examined for soft tissue alterations using a variation of the adaptation of Wilson's sectioning technique. These fetuses were fixed in Bouin's solution and sections retained in alcohol. The remaining fetuses were evicerated, cleared, stained with alizarin red S and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal preparations were retained in glycerin with thymol added.
Statistics:
Clinical observations and other proportional data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., body weights, body weight changes, feed consumption values, and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett’s Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p≤0.05), Dunnett’s Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Bartlett’s Test was significant (p≤0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p≤0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher’s Exact Test was used to analyze the data. Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Indices:
Maternal absolute and relative feed consumption.

Litter observations included fetal weights (male and female) and counts of corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, and resorbed conceptuses.
Historical control data:
Historical control data was available from this laboratory for the period from January 2005 to January 2007.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All female rats survived until scheduled sacrifice and there were no clinical observations that were considered related to test article administration. There were no gross lesions reported.

Weight losses were recorded on the first 2 days of dosing at the 1000 mg/kg bwt/day dose level, resulting in 5% reductions over control values. Overall weight gain was 5% less than the vehicle control group for the entire dosage period (days 7 to 18). Reductions in maternal weight gains were not statistically significant (Table 2).

Reductions in body weight gains were associated with significant reductions in absolute and relative feed consumption values at the 1000 mg/kg bwt/day dose level (Table 3). These occurred on gestation days 7 to 10, 10 to 12 and for the entire dosage period as compared with vehicle controls.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOEL
Effect level:
500 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:
The 1000 mg/kg bwt/day dose level represented an apparent threshold for developmental toxicity as evidenced by an approximately 3% reduction in fetal body and an associated retardation in ossification of the metatarsal bones. These variations were within historical ranges and were not considered of toxicological significance.

Reflecting the slight maternal toxicity noted, there were increases in supernumerary thoracic ribs, with associated increases and decreases in thoracic and lumbar vertebrae, respectively. These variations are reversible and are often observed at maternally toxic doses (Table 4).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: Developmental toxicity - Minimal reductions in foetal weights and small increase in reversible variations in skeletal

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 2: MATERNAL BODY WEIGHT GAINS - SUMMARY

DOSE GROUP

I

II

III

IV

DOSAGE (mg/kg bwt/day)

0 (control)

250

500

1000

RATS TESTED

N

25

25

25

25

PREGNANT

N

22

24

24

25

MATERNAL BODY

WEIGHT CHANGE (G)

DAYS 0 - 7

MEAN±S.D.

+42.5 ± 7.2

+42.0 ± 8.2

+40.7 ± 10.6

+39.8 ± 7.5

DAYS 7 - 8

MEAN±S.D.

-2.6 ± 4.6

-0.4 ± 5.2

-1.2 ± 5.5

-4.1 ± 6.8

DAYS 8 - 9

MEAN±S.D.

+1.4 ± 4.3

+2.0 ± 5.6

+1.5 ± 4.0

-1.1 ± 6.2

DAYS 9 - 10

MEAN±S.D.

+3.2 ± 4.6

+4.0 ± 4.8

+6.1 ± 4.2

+5.4 ± 5.7

DAYS 7 - 10

MEAN±S.D.

+2.0 ± 6.5

+5.5 ± 7.0

+6.4 ± 8.6

+0.1 ± 9.4

DAYS 10 - 12

MEAN±S.D.

+12.0 ± 5.3

+14.8 ± 5.4

+12.9 ± 5.2

+11.8 ± 9.6

DAYS 12 - 15

MEAN±S.D.

+17.7 ± 8.5

+17.1 ± 9.5

+19.8 ± 5.8

+18.4 ± 7.3

DAYS 15 - 18

MEAN±S.D.

+41.2 ± 9.6

+39.4 ± 8.8

+41.2 ± 7.3

+39.0 ± 7.7

DAYS 7 - 18

MEAN±S.D.

+73.0 ± 17.0

+76.8 ± 15.2

+80.2 ± 14.5

+69.3 ± 15.1

DAYS 18 - 21

MEAN±S.D.

+59.4 ± 11.4

+54.2 ± 8.7

+61.0 ± 10.8

+58.1 ± 12.8

DAYS 7 - 21

MEAN±S.D.

+132.4 ± 22.6

+130.9 ± 16.7

+141.2 ± 20.5

+127.4 ± 20.5

DAYS 0 - 21

MEAN±S.D.

+174.9 ± 25.6

+173.0 ± 21.0

+181.9 ± 27.2

+167.2 ± 23.6

Table 3: MATERNAL RELATIVE FEED CONSUMPTION VALUES (G/KG/DAY) - SUMMARY

DOSAGE GROUP

I

II

III

IV

DOSAGE (MG/KG/DAY)a

0 (VEHICLE)

250

500

1000

RATS TESTED

N

25

25

25

25

PREGNANT

N

22

24

24

25

MATERNAL FEED

CONSUMPTION (G/KG/DAY)

DAYS 0 - 7

MEAN±S.D.

96.2 ± 6.8

94.2 ± 6.9

93.5 ± 10.6

92.5 ± 6.5

[ 23]b

[ 23]b

DAYS 7 - 10

MEAN±S.D.

58.6 ± 10.2

60.8 ± 9.7

60.4 ± 12.7

46.2 ± 11.3**

[ 23]b

DAYS 10 - 12

MEAN±S.D.

61.4 ± 9.6

65.4 ± 8.8

60.0 ± 7.9

54.2 ± 15.5*

DAYS 12 - 15

MEAN±S.D.

59.8 ± 7.2

63.5 ± 9.7

59.6 ± 7.1

54.9 ± 9.3

[ 22]b

[ 23]b

DAYS 15 - 18

MEAN±S.D.

63.8 ± 14.0

68.7 ± 7.6

64.2 ± 10.0

60.8 ± 8.9

[ 21]b

DAYS 7 - 18

MEAN±S.D.

61.0 ± 5.6

64.5 ± 6.0

61.4 ± 7.3

54.1 ± 6.1**

[ 21]b

DAYS 18 - 21

MEAN±S.D.

73.1 ± 8.1

71.8 ± 8.7

74.6 ± 7.1

76.2 ± 8.1

DAYS 7 - 21

MEAN±S.D.

63.7 ± 4.8

66.4 ± 5.4

64.7 ± 6.0

59.6 ± 5.3*

DAYS 0 - 21

MEAN±S.D.

69.5 ± 4.0

70.5 ± 4.6

69.0 ± 5.3

66.0 ± 3.8*

[ ] = NUMBER OF VALUES AVERAGED

a. Dosage occurred on days 7 through 17 of gestation.

b. Excludes values that could not be calculated or appeared incorrectly recorded, as well as those associated with spillage.

* Significantly different from the vehicle control group value (p≤0.05). ** Significantly different from the vehicle control group value (p≤0.01).

Table 4: Dosage-Dependent Statistically Significant Fetal Observations (Average Ossification Sites per Fetus per Litter)

Observation

Dosage Group (DGs 7 through 17)

mg/kg/day

0 (Vehicle)

250

500

1000

Historical Controla

Litters

22

24

24

25

982

Fetuses

155

164

183

184

7305

Rats tested, n

25

25

25

25

Rats pregnant, n (%)

22 (88)

24 (96)

24 (96)

25 (100)

Corpora lutea, mean ± SD

14.6 ± 2.4

14.9 ± 2.8

16.3 ± 2.4

15.8 ± 2.3

Implantations, mean ± SD

14.0 ± 2.1

13.8 ± 2.8

15.2 ± 1.7

14.8 ± 1.8

Resorptions, mean ± SD

0.6 ± 0.7

0.8 ± 0.9

0.6 ± 0.6

0.5 ± 1.2

Litter size, mean ± SD

13.4 ± 2.2

13.0 ± 2.4

14.6 ± 2.0

14.3 ± 2.4

Fetal body weight/litter, mean ± SD

5.50 ± 0.28

5.54 ± 0.30

5.55 ± 0.31

5.33 ± 0.33

Male fetuses

5.63 ± 0.28

5.73 ± 0.31

5.68 ± 0.28

5.45 ± 0.36

Female fetuses

5.38 ± 0.29

5.37 ± 0.28

5.42 ± 0.35

5.18 ± 0.32*

Live fetuses/group

295

311

350

358

Dead fetuses/group

0

0

0

0

Ribs (pairs)b,c

13.06 ± 0.11

13.06 ± 0.10

13.09 ± 0.13

13.19 ± 0.23d

13.06 (13.02-13.12)

Thoracic Vertebraeb,c

13.08 ± 0.15

13.08 ± 0.16

13.12 ± 0.17

13.24 ± 0.28*,d

13.08 (13.02-13.16)

Lumbar Vertebraeb,c

5.92 ± 0.15

5.91 ± 0.16

5.87 ± 0.17

5.75 ± 0.27**,d

5.92 (5.83-5.97)

Metatarsalsb,c

4.95 ± 0.12

4.87 ± 0.21

4.91 ± 0.12

4.78 ± 0.27**,d

4.83 (4.68-4.95)

a. Studies (N = 44) conducted at the Testing Facility between January 2005 and January 2007 in rats of the same strain.

b. Means and Standard Deviations

c. Mean and range of means per study

d. Bold = effect of test article because values were significant and/or exceeded historical ranges. DGs = Days of Gestation

* = Significantly different from the vehicle control group value (p≤0.05). ** = Significantly different from the vehicle control group value (p≤0.01).

Testing facility historical control data for mean fetal body weight ranges (82 studies): combined male/female, 4.94 to 5.93 g; male fetuses, 5.10 to 5.98 g; female fetuses, 4.80 to 5.78 g.

Applicant's summary and conclusion

Conclusions:
On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) was 1000 mg/kg bwt/day. At this dose level, maternal body weight gains were reduced (not significant) and feed consumption was significantly reduced. The 500 mg/kg bwt/day dose level was assigned as the NOEL for maternal effects.

The developmental NOAEL was also 1000 mg/kg bwt/day. This was based on minimal reductions in female fetal weights and small increases in reversible variations in skeletal ossification. The 500 mg/kg bwt/day dose level was assigned as the NOEL for developmental effects.
Executive summary:

Dimyrcetol was administered in corn oil by gavage to groups of pregnant Sprague-Dawley rats at dosages of 0, 250, 500 or 1000 mg/kg bwt/day on gestational days 7 to 17. Viability, clinical signs, body weights and feed consumption were observed or measured for all animals. Animals were sacrificed on gestational day 21 and Caesarean sectioning performed. Fetuses were weighed and examined for sex, gross changes, and soft tissue or skeletal alterations. No clinical signs attributable to dimyrcetol administration were observed. At the 1000 mg/kg bwt/day dose level, mean maternal body weight gains were reduced 5% versus controls. Absolute and relative feed consumption were also reduced at this dose level. The minimal maternal effects noted were associated with minimal (approx. 3%) reductions in fetal body weights, reversible variations in ossification and increases in supernumerary thoracic ribs with associated increases or decreases in thoracic and lumbar vertebrae, respectively. Findings of delayed skeletal ossification are generally considered readily reversible (Carney and Kimmel, 2007 "Interpretation of skeletal variations for human risk assessment: Delayed ossification and wavy ribs", Birth Defects Research 80:473 -496). The minimal developmental delays (reduced pup weights, delayed ossificaiton, changes in metatarsals) are all expected to be readily reversible based on studies that have investigated the progression and outcome of similar effects.