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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:
13 April 2016 - 28 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

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

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
- Appearance: Yellow viscous liquid
- Storage conditions: At room temperature protected from light
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the vehicle: Stability for at least 24 hours at room temperature protected from light, for at least 8 days in the refrigerator and for at least 3 weeks in the freezer (≤-15°C) is confirmed over the concentration range 1 to 200 mg/mL.

Test animals

Species:
rat
Strain:
other: Crl:WI (Han), outbred, SPF-quality
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation, g: mean (SD), day 2 post-coitum: controls: 209 (14.3), low-dose: 209 (14.2), mid-dose 210 (10.6), high dose 208 (17.1)
- Fasting period before study: no
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to treatment

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

IN-LIFE DATES: From: 10 July 2016 To: 28 July 2016 (main study)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for specific gravity of the test item (1.15) and vehicle (1.125). No correction was made for the purity/composition of the test item.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 1.5, 5 and 15 mg/mL
- Amount of vehicle: 5 mL/kg bw. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (12 July 2016), according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Analysis was based on the analytical method validated for the test item and was conducted by a UPLC method.
Details on mating procedure:
- Impregnation procedure: not specified, performed at the supplier. The animals were at Day 0 or 1 post-coitum on arrival at the Test Facility.
- Proof of pregnancy: [vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
From days 6 to day 20 post-coitum, inclusive
Frequency of treatment:
Once dayly for 7 days/week
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle controls
Dose / conc.:
7.5 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected based on results of the dose range finding study.
- Rationale for animal assignment: 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: at least twice daily for mortality and viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from Day 2 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION : Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: thoracic and abdominal examination, with special attention being paid to the reproductive organs. Furhtermore the livers of all animals were dissected and examined.
Each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths (early and late resorptions).
- The weight of each fetus
- The sex of each fetus from the ano-genital distance (during necropsy) and also from
gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
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: 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 for the comparison of the treated groups and the control group.
- 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 post-implantation 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. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.
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:
Preimplantation loss (%) = ((number of corpora lutea - number of implantation)/number of corpora lutea) x 100
Post-implantation loss (%) = ((number of implantation sites - number of live fetuses)/number of implantation sites) x 100
Viable fetuses affected/litter (%) = (number of viable fetuses affted/litter / number of viable fetuses/litter) x 100
Historical control data:
Historical control data for rats of this strain from the testing laboratory are available from 2014-2015.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Incidental findings that were noted included alopecia, salivation and piloerection. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed and in absence of a dose related-trend, these findings were not considered to be treatment related.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 75 mg/kg bw/day, mean body weights were slightly lower than controls from Day 9 post-coitum onwards. This change was not statistically significant. Mean body weight gain was statistically significantly lower at 25 mg/kg bw/day on Day 12 post-coitum, and at 75 mg/kg bw/day on Days 9-18 post-coitum when compared to controls.
Body weight gain corrected for gravid uterus was statistically significantly lower at 75 mg/kg bw/day when compared to controls.
No toxicologically relevant changes in body weights, body weight gain or weight gain corrected for gravid uterus were noted by treatment at 7.5 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative food consumption were significantly reduced at 75 mg/kg bw/day from Day 6 post-coitum onwards (not statistically significant for relative food consumption on Days 15-18 post-coitum). At 25 mg/kg bw/day, absolute and relative food consumption were statistically significantly reduced on Days 6-12 post-coitum and on Days 6-9 post-coitum, respectively.
Food consumption before or after correction for body weight at 7.5 mg/kg bw/day were comparable to control levels
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weights (both absolute and relative to terminal body weights) were statistically significantly increased at 75 mg/kg bw/day. An increase of 15% was noted for relative liver weights. No toxicologically relevant changes in liver weights were noted at 7.5 and 25 mg/kg bw/day.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related non-adverse microscopic findings were noted in the liver of the 75 mg/kg bw/day group. An increased glycogen content was present in the liver at 75 mg/kg bw/day up to slight degree. This was considered to be within the range of background pathology encountered in pregnant non-fasted female rats of this age and strain and for this study type and therefore non-adverse.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In the 75 mg/kg bw/day group, a left lateral liver lobe grown together with the stomach and a diaphragmatic hernia of the right median liver lobe were noted for single females. Moreover, one control female showed a discolored thymus and one control female showed alopecia, confirming the clinical sign observed during the in-life phase. At the incidence observed, these findings were considered to be incidental and not toxicologically relevant.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on pre- or post-implantation loss by treatment up to 75 mg/kg bw/day. Although (not statistically significant) higher number of pre-implanation losses was observed in high-dose group (10.9% vs 4.5% in controls), it still fell within the historical control values.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on the number of litters lost by resorption by treatment up to and including 75 mg/kg bw/day.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on the number of early or late resorptions by treatment up to and including 75 mg/kg bw/day.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on the number of dead fetuses by treatment up to and including 75 mg/kg bw/day.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on the pregnancy duration by treatment up to and including 75 mg/kg bw/day/
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There were no effects on pregnancy duration.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females were pregnant and had litters with viable fetuses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to and including 75 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.3 and 5.1 gram for the control, 7.5, 25 and 75 mg/kg bw/day groups, respectively
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no toxicologically relevant effects on fetal body weights (both sexes) noted by treatment up to 75 mg/kg bw/day.
Mean combined (male and female) fetal body weights were 5.3, 5.3, 5.3 and 5.1 gram for the control, 7.5, 25 and 75 mg/kg bw/day groups, respectively.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no reductions in numbers of live offspring at any dose level.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to and including 75 mg/kg bw/day.
Mean sex ratios (males:females) were 54:46, 57: 43, 58:42 and 50:50 for the control, 7.5, 25 and 75 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.5, 11.2, 10.9 and 11.0 fetuses/litter for the control, 7.5, 25 and 75 mg/kg bw/day groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to and including 75 mg/kg bw/day.
The only external malformation that occurred among fetuses in this study was observed in Group 3 fetus. This fetus had an inwards rotated hind limb whereby no structural abnormality could be detected skeletally and as it occurred singly, it was considered to be a chance finding.
In addition, a late resorption with generalized edema (anasarca) was noted in the control group and as such considered toxicologically irrelevant.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to and including 75 mg/kg bw/day.
Malformations that were revealed at skeletal examination included in Group 4 severely malaligned sternebrae and vertebral anomaly without associated rib anomaly, and in Group 3 bent scapula and humerus. No malformations were observed in Group 2 and in the control Group, one fetus had a vertebral anomaly with associated rib anomaly. The single occurrence and group distribution of these malformations do not suggest any treatment relationship. Moreover, all were previously seen in historical controls.
Skeletal variations occurred at an incidence of 79.5%, 82.4%, 84.1% and 82.6% per litter in Groups 1, 2, 3 and 4, respectively. All variations noted were not considered treatment related as they occurred in the absence of a dose-dependent relationship, infrequently and/or at frequencies that were within the range of available historical control data.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to and including 75 mg/kg bwday.
Visceral malformations observed in groups treated with test item were a combination of interrupted aortic arch and atrial septum defect in one Group 4 fetus and a small eye in one Group 3 and one Group 4 fetuses. As these malformations occurred singly and there even was a control fetus that missed an eye, these were considered chance findings.
Remaining visceral malformations occurred in a control fetus that had situs inversus and abnormal lung lobation. The variations in this study occurred at low incidences, in the absence of a dose-related incidence trend or were observed in a control fetus only. Therefore, these visceral malformations and variations were not considered to be treatment related.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed at the highest tested dose

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Accuracy and homogeneity

The concentrations analysed in the formulations of Group 2 and Group 3 were in agreement with target concentrations (i.e. mean accuracies 91% for group 2 (n = 6) and 92% for group 3 (n = 2)).

The mean accuracy of the formulation of Group 4 was slightly below the target concentration (i.e. 89% of target). The accuracy of preparation of the formulation is considered acceptable

since the deviation is small and in line with the results of the QC samples at the similar concentration level of 15 mg/g.

The formulations of Group 2 and Group 4 were homogeneous (coefficient of variation 0.86% for group 2 and 2.6% for group 4, n= 6).

Table 1. Summary of developmental effects in the study.

Dose level (mg/kg bw/day)

0

8

25

75

Pregnant/total dams

22/22

22/22

22/22

22/22

-early resorptions

-late resorptions

(% per litter)

8.1

0.4

2.7

0.0

4.1

0.0

5.0

0.0

Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses only

0

0

0

0

Pre-implantation loss (number and percent)

13 (4.5%)

26 (8.5%)

27 (8.9%)

34 (10.9%)

Post-implantation loss (number and percent)

17 (8.5%)

7 (2.7%)

11 (4.1%)

13 (5.0%)

Body weight on day 21 (g)

332

335

327

320

Body weight gain day 6-21 (%)

46

48

44

42

Gravid uterine weight (g)

74.6

77.3

75.4

74.0

Mean live offspring (number)

10.5

11.2

10.9

11.0

Live offspring (percent)

91.5

97.3

95.9

95.0

Mean fetal/pup body weight males (g)

5.4

5.3

5.4

5.2

Mean fetal body weight females

5.1

5.1

5.2

5.0

Mean fetal body weight (sexes combined)

5.3

5.3

5.3

5.1

Malformations (including runts) number and percent of fetuses per litter

 

 

3 (2.5%)

 

 

0 (0.0%)

 

 

3 (2.3%)

 

 

4 (3.2%)

Variations (% per litter)

-external

-soft tissue

-skeletal

 

0

8.3

79.5

 

0

3.8

82.4

 

0

13.5

97.6

 

0

5.0

87.6

Table 2. Historical control data of fetal examinations

2-AMYLANTHRAQUINONE

 

 

 

 

 

 

Project 511244

 

 

 

 

 

 

 

 

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)

 

 

 

 

 

 

 

Gestation Day 21

 

Mean of Study Means

 

 

 

 

 

Study Date Range: 2014 - 2015

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Endpoint

Total

Mean

SD

Median

Min

Max

P5

P95

No of Studies

13

 

 

 

 

 

 

 

Total No. of Animals in the Control Group

304

 

 

 

 

 

 

 

No. of Animals that Died

0

 

 

 

 

 

 

 

No. of Animals that were Euthanized

0

 

 

 

 

 

 

 

No. of Animals that Aborted or Delivered

3

 

 

 

 

 

 

 

Percent Pregnant

 

98.8

2.73

100.0

90.9

100.0

97.1

100.0

No. of Animals Examined at Laparohysterectomy

301

 

 

 

 

 

 

 

No. Nongravid

4

 

 

 

 

 

 

 

No. Gravid

297

 

 

 

 

 

 

 

No. with Only Resorptions

2

 

 

 

 

 

 

 

No. of Dams with Live Fetuses

295

 

 

 

 

 

 

 

Mean No. Viable Fetuses/Dam

 

10.7

0.71

10.6

9.1

11.6

10.3

11.2

Total No. Viable Fetuses

3194

 

 

 

 

 

 

 

Viable Fetuses (%/Litter)

 

95.2

2.63

95.9

88.9

98.4

93.6

96.8

Mean No. Postimplantation Loss/Dam

 

0.5

0.15

0.4

0.2

0.7

0.4

0.6

Total No. Postimplantation Losses

134

 

 

 

 

 

 

 

Postimplantation Loss (%/Litter)

 

4.8

2.63

4.1

1.6

11.1

3.2

6.4

Dead Fetuses (%/Litter)

 

0.0

0.11

0.0

0.0

0.4

0.0

0.1

Early Resorptions (%/Litter)

 

4.7

2.62

4.1

1.6

11.1

3.2

6.3

Late Resorptions (%/Litter)

 

0.0

0.11

0.0

0.0

0.4

0.0

0.1

Mean No. Implantations/Dam

 

11.2

0.69

11.1

9.6

12.0

10.8

11.6

Mean No. Corpora Lutea/Dam

 

11.9

0.71

11.7

10.9

13.2

11.5

12.3

Mean No. Preimplantation Loss/Dam

 

0.7

0.32

0.6

0.2

1.3

0.5

0.9

Total No. Preimplantation Losses

207

 

 

 

 

 

 

 

Preimplantation Loss (%/Litter)

 

6.2

3.43

5.8

2.0

14.5

4.2

8.3

Total No. Male Fetuses

1617

 

 

 

 

 

 

 

Total No. Female Fetuses

1577

 

 

 

 

 

 

 

% Males/Litter

 

50.8

2.12

50.7

46.6

53.7

49.5

52.0

% Female/Litter

 

49.2

2.12

49.3

46.3

53.4

48.0

50.5

Mean Fetal Body Weight (g)

 

5.2

0.08

5.2

5.1

5.3

5.1

5.2

Mean Male Body Weight (g)

 

5.4

0.10

5.4

5.2

5.5

5.3

5.4

Mean Female Body Weight (g)

 

5.1

0.07

5.1

5.0

5.2

5.0

5.1

Mean Male Placenta Weight (g)1

 

0.46

0.01

0.47

0.44

0.47

0.4

0.5

Mean Female Placenta Weight (g)1

 

0.44

0.01

0.44

0.42

0.45

0.4

0.5

1Based on 4 datasets

2-AMYLANTHRAQUINONE

 

 

 

 

 

 

 

Project 511244

APPENDIX 5

 

 

 

 

 

 

 

 

 

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)

 

 

 

 

 

 

 

 

 

Gestation Day 21

 

 

 

 

 

 

 

 

 

Study Date Range: 2014 - 2015

 

 

 

 

 

 

 

 

 

No. of Studies

13

 

 

 

 

 

 

 

 

Total No. Fetuses/Litters Examined Externally

3194

295

 

 

 

 

 

 

 

Total No. Fetuses/Litters Examined Viscerally

2061

295

 

 

 

 

 

 

 

Total No. Fetuses/Litters Examined Skeletally

2059

295

 

 

 

 

 

 

 

 

Mean of Study Means

 

 

 

 

 

Summary Incidence

 

(% Per Litter Basis)

 

 

 

 

 

(Total No. Affected)

MALFORMATIONS

Mean

SD

Median

Min

Max

P5

P95

Fetuses

Litters

Total External Malformations

 

 

 

 

 

 

 

1

1

Total Visceral Malformations

 

 

 

 

 

 

 

7

7

Total Skeletal Malformations

 

 

 

 

 

 

 

15

15

Total Malformations

 

 

 

 

 

 

 

22

22

EXTERNAL

 

 

 

 

 

 

 

 

 

Exencephaly

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

Eye(s)- Open

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

VISCERAL

 

 

 

 

 

 

 

 

 

Diaphragmatic Hernia

0.0

0.08

0.0

0.0

0.3

0.0

0.1

1

1

Eye(s)- Absent and/or Small

0.1

0.26

0.0

0.0

0.9

0.0

0.2

3

3

Hydrocephaly- External

0.0

0.12

0.0

0.0

0.5

0.0

0.1

1

1

Situs Inversus

0.2

0.34

0.0

0.0

1.0

0.0

0.4

3

3

SKELETAL

 

 

 

 

 

 

 

 

 

Jaw- Upper Jaw Small

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

Jaw- Lower Jaw Absent or Small

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

Limb Bone(s)- Bent

0.3

0.44

0.0

0.0

1.1

0.0

0.5

4

4

Rib Anomaly

0.1

0.31

0.0

0.0

1.1

0.0

0.3

1

1

Skull Anomaly

0.1

0.34

0.0

0.0

1.2

0.0

0.3

2

2

Sternebra(e)- Fused

0.1

0.29

0.0

0.0

1.0

0.0

0.3

2

2

Sternebra(e) Malaligned (Severe)

0.0

0.08

0.0

0.0

0.3

0.0

0.1

1

1

Sternoschisis

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

Vertebral Anomaly With or Without Associated Rib Anomaly

0.2

0.53

0.0

0.0

1.9

0.0

0.5

3

3

Vertebral Centra Anomaly

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

2-AMYLANTHRAQUINONE

 

 

 

 

 

 

 

Project 511244

 

 

 

 

 

 

 

 

 

Historical Control Data Rat: Crl:WI(Han) (outbred. SPF-Quality)

 

 

 

 

 

 

 

 

 

Gestation Day 21

 

 

 

 

 

 

 

 

 

 

Mean of Study Means

 

 

 

 

Summary Incidence

 

(% Per Litter Basis)

 

 

 

 

 

(Total No. Affected)

VARIATIONS

Mean

SD

Median

Min

Max

P5

P95

Fetuses

Litters

EXTERNAL

 

 

 

 

 

 

 

 

 

None Observed

 

 

 

 

 

 

 

 

 

VISCERAL

 

 

 

 

 

 

 

 

 

Kidney(s)- Renal Papilla(e) Absent and/or Small

0.1

0.25

0.0

0.0

0.9

0.0

0.2

2

2

Liver- Appendix

1.2

0.56

1.3

0.3

2.3

0.9

1.6

23

21

Liver- Discolored

0.1

0.30

0.0

0.0

1.0

0.0

0.3

3

3

Liver- Small Supernumerary Lobe(s)

4.0

1.96

4.0

1.3

7.7

2.8

5.2

69

58

Spleen- Supernumerary

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

Thymus- Partially Undescended Horn(s)

1.3

1.55

0.8

0.0

4.3

0.3

2.2

34

23

Thyroid- Discolored

0.1

0.36

0.0

0.0

1.3

0.0

0.3

1

1

Ureter(s)- Convoluted

1.0

2.39

0.0

0.0

8.7

0.0

2.5

43

28

Ureter(s)- Dilated

0.9

2.33

0.0

0.0

8.5

0.0

2.3

44

19

SKELETAL

 

 

 

 

 

 

 

 

 

7th Cervical Rudimentary Rib(s)

1.7

1.34

1.2

0.0

4.4

0.9

2.5

30

26

7th Cervical Full Rib(s)

0.1

0.36

0.0

0.0

1.1

0.0

0.4

2

2

14th Full Rib(s)

5.7

4.65

5.2

0.0

13.1

2.9

8.5

88

64

14th Rudimentary Rib(s)

44.1

19.84

54.4

19.0

72.0

32.1

56.1

798

250

Metacarpal(s) and/or Metatarsal(s) Unossified

2.2

1.97

1.0

0.0

6.3

1.0

3.4

41

24

Pelvic Girdle- Caudal Shift

6.6

3.77

7.1

1.7

12.8

4.3

8.9

127

71

Rib(s)- Bent

10.6

7.78

10.2

0.8

22.3

5.9

15.3

162

85

Rib(s)- Short

0.0

0.06

0.0

0.0

0.2

0.0

0.0

1

1

Skull- Reduced Ossification

2.7

2.55

1.8

0.0

7.0

1.2

4.3

81

46

Skull- Supernumerary Site

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

Sternebra(e) #1, #2, #3 and/or #4 Unossified

0.2

0.31

0.0

0.0

0.8

0.0

0.3

3

3

Sternebra(e) #5 and/or #6 Unossified

0.9

1.33

0.0

0.0

4.1

0.1

1.7

37

23

Sternebrae- Malaligned (Slight or Moderate)

11.1

5.72

8.9

4.4

21.3

7.6

14.5

188

131

Sternum- Supernumerary Ossification Site

0.1

0.31

0.0

0.0

1.1

0.0

0.3

1

1

Vertebral Centra- Reduced Ossification

0.6

0.88

0.4

0.0

3.0

0.1

1.2

12

12

Applicant's summary and conclusion

Conclusions:
In a developmental toxicity study with rats, oral exposure to the test substance 2-amylanthraquinone during days 6-20 of gestation did not induce any developmental effects up to and including the top dose of 75 mg/kg bw/day. The NOAEL for maternal toxicity was 25 mg/kg bw/day, based on the changes in body weights and food consumption, as well as liver weight increase at the highest dose level.
Executive summary:

In a GLP-compliant OECD Guideline 414 study, the test substance 2 -amylanthraquinone was administered to pregnant rats by oral gavage during days 6 -20 of gestation. Maternal toxicity was observed at the highest dose level of 75 mg/kg bw/day, manifested in slightly lower body weights and statistically significantly lower body weight gain during the treatment period, as well as reduced food consumption. Relative liver weights were statistically significantly increased by 15% at 75 mg/kg bw/day compared to the concurrent vehicle controls. Based on these observations the NOAEL for maternal toxicity was set at the mid-dose of 25 mg/kg bw/day.

No developmental toxicity was observed at any dose level. There were also no toxicologically relevant effects on the numbers of pregnant females, corpora lutea and implantation sites, or in pre- or post-implantation loss by treatment up to and including the dose of 75 mg/kg bw/day. Based on this, the NOAEL for developmental toxicity was set at 75 mg/kg bw/day, the highest dose tested.