Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 November 2015 to 4 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
equivalent or similar to
Guideline:
other: - OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment, 24th July 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: para-Menthane
Chemical name Cyclohexane,1-methyl-4-(1-methylethyl)/1-isopropyl-4-methylcyclohexane
Appearance: colourless liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd. Han: WIST Rats
- Source: TOXI-COOP ZRT. 1103 Budapest, Cserkesz u. 90.
- Age at study initiation: females 10-11 weeks (males for mating 11-12 weeks)
- Weight at study initiation: 166-217 g
- Fasting period before study: none
- Housing: pre-mating period: 1-3 females /cage, 2 males/cage; during mating hours:1 male with 1- 3 females; during pregnancy: 2-3 sperm positive females /cage
- Diet: ssniff® SM R/M-Z+H complete diet (ssniff Spezialdiäten GmbH, D-59494 Soest Germany) ad libitum
- Water : tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22 °C
- Humidity (%): 34-47%
- Air changes (per hr): >10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: test item formulations was made with a frequency of up to 7 days, using a magnetic stirrer and stored at room temperature

VEHICLE: cornoil
- Justification for use and choice of vehicle (if other than water): low solubility of the test item in water
- Concentration test substance in vehicle: 0, 50, 150 and 500 mg/mL
- Dosing volume (if gavage): 2 mL
- Lot/batch no.: MKBV2080V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Five samples were taken from different places in the dosing formulation on day 7 and 18 of the study. Samples were diluted with n-hexane in duplicate except for the control. A volume of 0.5 mL of the internal standard solution was added before the samples were filled up to 10 mL final volume. Samples were stored overnight and analysed the next day.

GC/FID
Column: Rtx-502.2, 60 m x 0.25 mm x 1.4 µm, No: 1023994
Temperature program: 125°C to 150°C at 2°C/min
2 min hold
150°C to 180°C at 20°C/min
2 min hold
Injector: 200°C
Detector: FID, 200°C
Carrier gas: nitrogen, 2 mL/min, constant flow
Split ratio: 20
Injection volume: 0.5 µL
Retention times: para-Menthane 13.5-13.6 and 14.4-14.5 min
1-Octanol: 16.9-17.0 min
Evaluation: Sum of the areas of para-Menthane peaks is calculated and divided by 1-octanol peak area. This ratio is used for the computations.

Linearity over 0.1 – 3 mg/mL (r2 >= 0.998); repeatability 0.6-1.0% (7 injections at 500 and 1 mg/L)
Recovery in corn oil: 96+/-4% at 1 mg/L; at 500 mg/L 95+/-5%
Stability over 14 days:101-104% (room temperature) 98-104% (at 5 ± 3 °C) measured at 1 and 500 mg/L in corn oil
LOQ: 0.1 mg/L
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male/1-3 females
- Length of cohabitation: 2-4 hours
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
day 5-19 of gestation
Frequency of treatment:
daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
measured concentration 94% of nominal
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
measured concentration 100-103% of nominal
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
measured concentration 96-100% of nominal
No. of animals per sex per dose:
22 sperm positive females/group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based on the results of a DRF test where the substance at the dose level of 1000 mg/kg bw/day caused clinical signs such as digging in the bedding and salivation but no effects on the body weight and food consumption data of the animals and the intrauterine development of the fetuses. The treatment at the dose levels of 80, 200 and 500 mg/kg bw/day caused no maternal or fetal effects (TOXI-COOP ZRT 590-410-0850).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: mortality/morbidity
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: pre-mating and on day 0, 3, 5, 8, 11, 14, 17 and 20 of gestation (in addition on day 20 corrected for gravid uterus weight)

FOOD CONSUMPTION: Yes
-Time schedule for examinations: day 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes gross necropsy
- Sacrifice on gestation day 20
- Organs examined: uterus with cervix and left ovary were weighed

OTHER: On gestation days 13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding which indicated the implantation of conceptuses
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 implantation sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live fetuses: Yes
- Fetal death: yes
Fetal examinations:
- Fetal viability: Yes
- Fetal and placenta weight: Yes
- Fetal gender: Yes (measurement of anogenital distance)
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (fixed in Sanomiva mixture before dissection)
- Skeletal examinations: Yes: half per litter (examined under dissection microscope after fixation in alcian-blue-acetic acid-ethanol mixture and in isopropanol, the skeletons were stained by KOH-Alizarin red-S method)
- Head examinations: Yes: half per litter (fetuses selected for visceral examination Wilson's technique)
Statistics:
Bartlett's homogeneity of variance test or one-way ANOVA, chi-quare test
positive findings : distribution analysis and if normal concomitant ANOVA (Duncan's Multiple Range test) or Kolmogorov-Smirnov test.
non-normal distributions: Kruskal-Wallis One-Way analysis of variance

Indices:
Pre-implantation loss
Post-implantation loss
Sex distribution
External abnormalities/litter
Visceral abnormalities/litter
Skeletal abnormalities/litter

calculated as %, group mean
Historical control data:
included in the report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There was no mortality and no clinical signs were recorded during the in-life phase.

The number of sperm positive females was 88 in the study. Tthe number of evaluated litters was 79 (20 both in the control and 100 mg/kg bw/day, 22 in the 300 and 17 in the 1000 mg/kg bw/day dose groups respectively).


(sum, %)
DESCRIPTION DOSES: control 100 300 1000 mg/kg bw/day
No. of
animals: 20 20 22 17
CLINICAL SYMPTOMS
- none N 20 20 22 17
% 100 100 100 100
NECROPSY FINDINGS
- no macroscopic alterations N 20 20 22 17
% 100 100 100 100
Mortality:
no mortality observed
Description (incidence):
There was no mortality and no clinical signs were recorded during the in-life phase.

DATA OF FEMALES MORTALITY(sum, %)

Dose groups mg/kg bw/day Control 100 300 1000
No of preg. fem. died 0 0 0 0
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight and corrected body weight (including corrected body weight gain) of the females was similar in all groups during the in-life phase.
From gestation day 5 to 8 the mean body weight gain of the dams in the 1000 mg/kg bw/day group was slightly but statistically significantly lower (p<0.01) versus control which was attributed to the treatment with the test item. The body weight gain of the dams in the 300 and 100 mg/kg bw/day group was at the current control level.
Detailed information about the body weight developments are given in the tables below or can be found in the attached tables in pdf format.
The slight but significant decrease in body weight gain during day 5-8 at 1000 mg/kg bw is considered incidental and not related to treatment



APPENDIX III SUMMARY OF BODY WEIGHT AND BODY WEIGHT GAIN OF DAMS
Body Weight DOSE GROUPS (mg/kg bw/day)
TIME Gestational days Control 100 300 1000
0 Mean 195,1 195,1 194 192,5
SD 12,66 10,04 10,85 9,04
n 20 20 22 17 NS
3 Mean 208,3 210,4 207,5 206,6
SD 14,44 10,17 11,85 10,32
n 20 20 22 17 NS
5 Mean 217,9 220 216,7 214,6
SD 15,58 13,36 14,48 10,87
n 20 20 22 17 NS
8 Mean 229,7 232,4 229,4 222,5
SD 17,06 14,93 16,75 12,3
n 20 20 22 17 NS
11 Mean 245,3 248,8 246,8 235,8
SD 19,21 18,31 18,78 12,71
n 20 20 22 17 NS
14 Mean 258,7 261,4 260,2 248,9
SD 20,65 19,01 19,61 13,16
n 20 20 22 17 NS
17 Mean 282,6 285,7 286 237,5
SD 23,24 21,47 21,98 14,09
n 20 20 22 17 NS
20 Mean 319,2 320,7 321,3 307,6
SD 28,44 26,72 23,28 12,72
n 20 20 22 17 NS
REMARKS :
NS = Not Significant
* = p < 0.05
** = p < 0.01
U = Mann-Whitney U - test Versus Control
DN = Duncan's multiple range test

Body weight gain DOSE GROUPS (mg/kg bw/day)
TIME Gestational days Control 100 300 1000
0-3 Mean 13,3 15,3 13,5 14,1
SD 3,16 5,03 3,11 3,93
n 20 20 20 17 NS
3-5 Mean 9,6 9,6 9,2 8,1
SD 3,17 3,72 4,18 2,44
n 20 20 22 17 NS
5-8 Mean 11,8 12,4 12,7 7,8
SD 2,76 3,87 3,71 2,72
n 20 20 22 17 **DN
8-11 Mean 15,6 16,5 17,4 12,6
SD 4,33 4,67 3,89 9,23
n 20 20 22 17 NS
11-14 Mean 13,4 12,6 13,4 24,5
SD 3,44 3,94 4,64 5,36
n 20 20 22 17 NS
14-17 Mean 23,9 24,3 25,8 24,5
SD 4,33 5,07 5,31 5,36
n 20 20 22 17 NS
17-20 Mean 36,7 35 35,3 34,2
SD 8,42 7,77 5,17 7,39
n 20 20 22 17 NS
0-20 Mean 124,2 125,6 127,3 115,1
SD 19,4 19,02 15,77 8,08
n 20 20 22 17 NS
REMARKS :
NS = Not Significant
* = p < 0.05
** = p < 0.01
U = Mann-Whitney U - test Versus Control
DN = Duncan's multiple range test

SUMMARY OF GRAVID UTERINE WEIGHT, CORRECTED BODY WEIGHT AND CORRECTED BODY WEIGHT GAIN OF DAMS
DOSE GROUPS (mg/kg bw/day) Control 100 300 1000
Gravid uterine weight (g) Mean 65,2 62,1 63,4 62,1
SD 10,67 8,87 9,79 10,22 NS
N 20 20 22 17
Corrected body weight (g) Mean 254 258,6 257,9 245,5
SD 19,59 22,32 20,36 17,46
N 20 20 22 17 NS
Corrected body weight gain (g) Mean 59 63,5 63,9 53
SD 10,9 15,15 12,83 13,01
N 20 20 22 17 NS

REMARKS : NS = Not Significant
* = p < 0.05 ** = p < 0.01
U = Mann-Whitney U - test Versus Control DN = Duncan's multiple range test
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the dams in the 1000 mg/kg bw/day group was slightly but statistically significantly lower (p<0.01) from gestational day 5 to 14 (-23%, -20% and -11% relative to controls between days 5 to 8, 8 to 11 and 11 to 14, respectively) which was considered to be due to the treatment with the test item.

APPENDIX V SUMMARY OF FOOD CONSUMPTION DATA OF DAMS
DOSE GROUPS (mg/kg bw/day)
TIME Gestational days Control 100 300 1000
0-3 Mean 18,7 18,5 17,9 17,8
SD 0,87 1,41 0,92 0,69
n 20 20 22* 17** U
3-5 Mean 20,8 21,3 20,4 20,2
SD 1,06 2,18 1,25 0,59
n 20 20* 22 17 U
5-8 Mean 21,2 21,2 20,6 16,3
SD 0,67 2,29 1,66 1,22
n 20 20 22 17** U
8-11 Mean 22,2 22,7 21,8 17,7
SD 1,12 2,89 1,37 1,12
n 20 20 22 17** U
11-14 Mean 23,2 23,4 23,5 20,7
SD 1,51 2,51 1,22 2,31
n 20 20 22 17** U
14-17 Mean 22,9 23,6 23,7 22
SD 1,57 2,81 1,43 2,17
n 20 20 22 17 NS
17-20 Mean 23 23,9 24,9 22,9
SD 1,62 2,82 1,5 2,43
n 20 20 22** 17 U
REMARKS : NS = Not Significant
* = p < 0.05 ** = p < 0.01
U = Mann-Whitney U - test Versus Control DN = Duncan's multiple range test
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:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The whole number of pre-implantation loss was statistically significantly higher in the 1000 mg/kg bw/day group according to the Chi square test but not if the mean percent was evaluated. Moreover the relative value of 12.2% was within the control background data range (8.2-21.4%). Hence this slight increase was considered to be unrelated from the treatment. There were no significant differences in the mean number of corpora lutea, implantations, viable fetuses and their sex distribution. There was no significant increase of early- and late embryonic death and post-implantation loss in the test item treated groups. Moreover, early embryonic death and post-implantation loss was statistically significantly lower in the 1000 mg/kg bw/day group according to the Chi square analysis.

APPENDIX VI/A INTRAUTERINE MORTALITY, VIABLE FETUSES, SEX DISTRIBUTION

GROUPS (mg/kg bw/day): Control 100 300 1000
NUMBER OF DAMS: 20 20 22 17

Total Intrauterine Mortality % Mean±SD: 15.0± 9.33 15.3± 9.89 14.4 ±13.76 15.3± 13.41 NS
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
At 1000 mg/kg bw post-implantation loss was decreased significantly (while pre-implantation loss was slightly, but not-significantly increased).

APPENDIX VI/A INTRAUTERINE MORTALITY, VIABLE FETUSES, SEX DISTRIBUTION

GROUPS (mg/kg bw/day): Control 100 300 1000
NUMBER OF DAMS: 20 20 22 17

Preimplantation Loss % Mean±SD: 6.3±6.42 6.3±8.02 7.7±10.32 12.2±15.20 NS
Postimplantation Loss % Mean±SD: 9.3± 7.53 9.5± 8.74 7.5 ±9.55 3.1± 4.85 * DN
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
APPENDIX VI/A INTRAUTERINE MORTALITY, VIABLE FETUSES, SEX DISTRIBUTION

GROUPS (mg/kg bw/day): Control 100 300 1000
NUMBER OF DAMS: 20 20 22 17
Early Embryonic Death % Mean±SD: 8.1±7.05 6.6±7.65 6.3±7.07 2.7±4.75 NS
Late Embryonic Death % Mean±SD: 1.1 ±3.72 2.5±4.24 1.2± 5.81 0.0± 0.00 NS
Dead fetuses:
no effects observed
Description (incidence and severity):
no treatment related effects

APPENDIX VI/A INTRAUTERINE MORTALITY, VIABLE FETUSES, SEX DISTRIBUTION

GROUPS (mg/kg bw/day): Control 100 300 1000
NUMBER OF DAMS: 20 20 22 17
Dead Fetuses % Mean±SD: 0.0± 0.00 0.4± 1.60 0.0± 0.00 0.5± 1.87 NS
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The lower pregnancy rate at 1000 mg/kg bw is unrelated to treatment (all females were sperm positive at treatment start)
Females evaluated were 20, 20, 22 and 17 at 0, 100, 300 and 1000 mg/kg bw respectively.

APPENDIX I PREGNANCY DATA OF FEMALES, MORTALITY, MALFORMATIONS (sum, %)

Dose groups Control 100 mg/kg bw/day 300 mg/kg bw/day 1000 mg/kg bw/day
Number of sperm positive females 22 22 22 22
Number of females with no implantation but corpora lutea 0 1 0 1
Number of females with no implantation and no corpora lutea 2 1 0 4
Number and percent of pregnant females (females with implantation) 20 91% 20 91% 22 100% 17 77%
Number of dams with 3 or less implantations 0 0 0 0
Number of dams with total intrauterine death (total postimplantation loss) 0 0 0 0
Number of pregnant females died 0 0 0 0
Number of evaluated litters 20 20 22 17
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
other: the effect on body weight (gain) due to the decreased food consumption is negligible
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no effects

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no significant difference in the body weight of the fetuses in the experimental groups. The placental weight was slightly but statistically significantly lower in the 100, 300 and 1000 mg/kg bw/day and the relative placental weight in the 300 and 1000 mg/kg bw/day group if compared to the control. The placental weight values were slightly below the historical control level (654.9-747.5) in the 300 and 1000 mg/kg bw/day groups and in case of female fetuses in the 100 mg/kg bw/day group. Considering that the relative placental weight values for combined sexes were within the range of the background data (186.9-234.4) and showed no dose related tendency, the lower placental and relative placental weight values were likely not a consequence of the treatment.

APPENDIX VIII LITTER MEANS OF FETAL AND PLACENTAL WEIGHT
DOSE GROUPS (mg/kg bw/day) Control 100 300 1000
M+F M F M+F M F M+F M F M+F M F
Fetal weight (g) Mean 3,5 3,5 3,4 3,4 3,4 3,3 3,5 3,5 3,4 3,4 3,4 3,3
SD 0,25 0,25 0,25 0,2 0,27 0,19 2,1 0,22 0,22 0,28 0,29 0,3
N 20 20 20 20 20 20 22 22 22 17 17 17
N S NS NS
Placental weight (mg) Mean 716,5 729,6 699 656,5 660,7 652,6 648,3 651,1 643,3 634,4 645,1 623,3
SD 67,54 64,86 76,09 44,96 51,62 46,72 47,22 65,63 48,78 62,66 58,29 69,64
N 20 20 20 20** 20** 20* 22** 22** 22** 17** 17** 17**
DN DN DN
Relative placental weight (mg/g) Mean 207,7 207,3 207,3 197,9 194,6 200,5 188,8 184,6 191,4 190,2 188,5 193,1
SD 20,61 21,06 21,06 17,5 19,03 18,45 16,68 19,91 18,16 21,79 22,62 23,91
N 20 20 20 20 20 20 22** 22** 22* 17** 17** 17*
DN DN DN

Remarks: M = Male
F = Female
NS = Not Significant
* = p < 0.05
** = p < 0.01
U = Mann-Whitney U - test Versus Control
DN = Duncan's multiple range test
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 was no significant difference in the body weight of the fetuses in the experimental groups. The placental weight was slightly but statistically significantly lower in the 100, 300 and 1000 mg/kg bw/day and the relative placental weight in the 300 and 1000 mg/kg bw/day group if compared to the control. The placental weight values were slightly below the historical control level (654.9-747.5) in the 300 and 1000 mg/kg bw/day groups and in case of female fetuses in the 100 mg/kg bw/day group. Considering that the relative placental weight values for combined sexes were within the range of the background data (186.9-234.4) (Appendix XXIV/A) and showed no dose related tendency, the lower placental and relative placental weight values were likely not a consequence of the treatment.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no significant differences in the mean sex distribution.

APPENDIX VI/A SEX DISTRIBUTION

GROUPS (mg/kg bw/day): Control 100 300 1000
NUMBER OF DAMS: 20 20 22 17
Male fetuses % Mean±SD: 56.5± 12.49 46.6± 15.91 47.1±15.87 53.1± 17.23 NS
Female fetuses % Mean±SD: 43.5± 12.49 53.4± 15.91 52.9± 15.87 46.9 ±17.23 NS

Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 231, 222, 249 and 195 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

Malformations
One fetus was found with an umbilical hernia and malrotated left hindlimb in the 100 mg/kg bw/day dose group. The flexion of the hindlimb was not confirmed at skeletal examination. Considering that this was a single fetus and in the low dose this was judged to have likely no relationship with the test item. Moreover umbilical hernia occurs sporadically with low incidence unrelated to the treatment according to the experience with this species in this laboratory which is in line with historical control data of another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993) (11). There were no external malformations recorded in the 300 and 1000 mg/kg bw/day groups.

Variations
The incidence of body weight retarded fetuses and litters (below 2.89 g for males and below 2.77 g for females) was statistically significantly (p<0.05) higher in the 1000 mg/kg bw/day group according to the Chi square test. There was no significance detected if the incidence of affected litters or the percentile litter means were evaluated and no clear dose relationship was observed. Hence this was considered to be unrelated from the treatment. There was no statistical significance indicated in the other groups.

Placental abnormalities
There were no treatment related placental abnormalities found during the external examination. Two placentas were fused in the control group.

APPENDIX VII/A RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
(percentile litter means and SD)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000
EXTERNAL EXAMINATION
Litters examined N 20 20 22 17
Fetuses examined N 231 222 249 195
Fetuses with abnormalities Mean 4,4 8 3,6 9,4
SD 10,2 13,18 7,83 14,55
Variation Mean 4,4 7,5 3,6 9,4
SD 10,2 13,29 7,83 14,55
Malformation Mean 0 0,5 0 0
SD 0 2,24 0 0
Retarded in body weight Mean 4,4 8 3,6 9,4
SD 10,2 11,78 7,83 14,55

Remarks: * = p < 0.05
** = p < 0.01
U = Mann-Whitney U - test Versus Control
DN = Duncan's multiple range test

APPENDIX VII/A RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
(sum, %)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000
EXTERNAL EXAMINATION
Litters examined N 20 20 22 17
Fetuses examined N 231 222 249 195
Fetuses with abnormalities N 9 17 9 18*
% 4 8 4 9
Litters N 6 9 6 8
% 30 45 27 47
Variation N 9 16 9 18*
% 4 7 4 9
Litters N 6 8 6 8
% 30 40 27 47
Malformation N 0 1 0 0
% 0 0 0 0
Litters N 0 1 0 0
% 0 5 0 0
Retarded in body weight N 9 17 9 18*
% 4 8 4 9
Litters N 6 9 6 8
% 30 45 27 47

Remarks:
* = p < 0.05; CH2
** = p < 0.01; CH2


APPENDIX IX TYPES OF EXTERNAL ABNORMALITIES
(sum, %)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000

Number of Dams N 20 20 22 17
Fetuses examined N 231 222 249 195
Fetuses with abnormalities N 9 17 9 18*
% 4 8 4 9
Litters N 6 9 6 8
% 30 45 27 47
Variation N 9 16 9 18*
% 4 7 4 9
Litters N 6 8 6 8
% 30 40 27 47
Malformation N 0 1 0 0
% 0 0 0 0
Litters N 0 1 0 0
% 0 5 0 0

FETAL VARIATIONS

Retarded in body weight N 9 17 9 18*
% 4 8 4 9
Litters N 6 9 6 8
% 30 45 27 47
Hydrops fetalis N 0 1 0 0
% 0 0 0 0
Litters N 0 1 0 0
% 0 5 0 0


FETAL MALFORMATIONS

Multiple malformed N 0 1 0 0
% 0 0 0 0
Litters N 0 1 0 0
% 0 5 0 0


Placental abnormalities

Fused N 1 0 0 0
% 0 0 0 0
Litters N 1 0 0 0
% 5 0 0 0
Remarks:
* = p < 0.05; CH2
** = p < 0.01; CH2
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 118, 113, 125 and 97 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

Malformations:
There were two malformations found in the control group, one fetus had bent scapula (unilateral) and one with a dumb-bell shaped cartilage of a thoracic vertebral centrum. Three malformed fetuses were found in the 100 mg/kg bw/day group. One of these fetuses had a split sternum (this was the same fetus with umbilical hernia at external examination), bifurcate rib was recorded for a second and short femur, tibia and fibula for a third one. There were no malformations found in the 300 mg/kg bw/day dose group. Abnormal articulation of the fibula with calcaneus and talus was recorded for one fetus in the 1000 mg/kg bw/day group.
Split sternum may occur without any test item relationship with a low incidence according to the laboratory’s Historical Control Data (Appendix XXIV/B) of Toxi-Coop Zrt. This is in line with historical control data of another strain of rats, i.e. CrL:CD(R) BR rats (Lang, 1993) by which also bifurcate (branched) ribs and hypoplastic long bones of the hind limbs may occur in control animals (11). Considering the low incidence (one single fetus in the high dose group) of these different type of malformations the occurrence was likely not attributed to the treatment.

Variations:
Retardation of the skull, incomplete ossification or lack of ossification of the skull bones, hyoid, sternebra, vertebrae and metacarpal/metatarsal, bipartite supraoccipital, as well as bipartite sternal bodies, wavy ribs, dumb-bell shaped and bipartite and asymmetric thoracic or lumbar centra (with or without a slightly dumb-bell shaped cartilage), unossified SII arches, asymmetric ossification of metacarpal/metatarsal, asymmetric sacral/lumbar pelvic articulation and incompletely or not ossified pubic were evaluated as variations during the skeletal examination.

The incidence of 3 or less ossified sternebra increased significantly (p<0.05) in the 1000 mg/kg bw/day dose group however stayed within the historical control range (0-8.16%) and was not attributed to an effect of the test item. There were no significant differences in the different type of variations among the experimental groups.


APPENDIX VII/A RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
(percentile litter means and SD)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000
SKELETAL EXAMINATION
Litters examined N 20 20 22 17
Fetuses examined N 118 113 125 97
Fetuses with abnormalities Mean 10 12,1 19,2 13,4
SD 16,58 12,29 22,09 16,79
Variation Mean 8,3 9,1 19,2 12,6
SD 15,76 11,17 2,09 17,1
Malformation Mean 1,7 2,9 0 0,8
SD 5,13 7,29 0 3,46

Remarks: * = p < 0.05
** = p < 0.01
U = Mann-Whitney U - test Versus Control
DN = Duncan's multiple range test


APPENDIX VII/A RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
(sum, %)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000
SKELETAL EXAMINATION
Litters examined N 20 20 22 17
Fetuses examined N 118 113 125 97
Fetuses with abnormalities N 11 14 24* 13
% 9 12 19 13
Litters N 7 11 12 9
% 35 55 55 53
Variation N 9 11 24** 12
% 8 10 19 12
Litters N 6 9 12 8
% 30 45 55 47
Malformation N 2 3 0 1
% 2 3 0 1
Litters N 2 3 0 1
% 10 15 0 6

Remarks:
* = p < 0.05; CH2
** = p < 0.01; CH2



APPENDIX XI TYPES OF SKELETAL ABNORMALITIES
(sum, %)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000

Number of Dams N 20 20 22 17
Fetuses examined N 118 113 125 97
Fetuses with abnormalities N 11 14 24* 13
% 9 12 19 13
Litters N 7 11 12 9
% 35 55 55 53
Variation N 9 11 24** 12
% 8 10 19 12
Litters N 6 9 12 8
% 30 45 47
Malformation N 2 3 0 1
% 2 3 0 1
Litters N 2 3 0 1
% 10 15 0 6

FETAL VARIATIONS
Skull
retarded N 0 1 0 0
% 0 1 0 0
Litters N 0 1 0 0
% 0 5 0 0
incomplete ossification (more than 3 bones) N 1 1 1 0
% 1 1 1 0
Litters N 1 1 1 0
% 5 5 5 0
incomplete ossification, marked (one bone or more) N 5 5 10 5
% 4 4 8 5
Litters N 4 3 6 3
% 20 15 27 18
supra occipital bipartite N 0 1 0 2
% 0 1 0 2
Litters N 0 1 0 2
% 0 5 0 12
hyoid not ossified N 0 0 0 1
% 0 0 0 1
Litters N 0 0 0 1
% 0 0 0 6
Sternebra
3 or less ossified N 1 3 3 6*
% 1 3 2 6
Litters N 1 2 3 5*
% 5 10 14 29
bipartite N 0 1 0 1
% 0 1 0 1
Litters N 0 1 0 1
% 0 5 0 6
Ribs
wavy N 5 0* 2 2
% 4 0 2 2
Litters N 3 0 2 2
% 15 0 9 12
Vertebrae
Thoracic centra
- dumb-bell shaped and/or asymmetric (more than 3) N 0 0 1 0
% 0 0 1 0
Litters N 0 0 1 0
% 0 0 5 0
bipartite or bipartite and asymmetric N 0 0 1 2
% 0 0 1 2
Litters N 0 0 1 2
% 0 0 5 12
dumb-bell shaped or bipartite N 1 1 1 1
and cartilage slightly dumb-bell shaped % 1 1 1 1
Litters N 1 1 1 1
% 5 5 5 6
Thoracic and lumbar centra and/or arches
not ossified (more than 3) N 0 0 0 1
% 0 0 0 1
Litters N 0 0 0 1
% 0 0 0 6

Lumbar/sacral arches
asymmetric pelvic articulation N 0 1 7** 1
% 0 1 6 1
Litters N 0 1 5* 1
% 0 5 23 6

SII arches N 0 0 1 0
not ossified % 0 0 1 0
Litters N 0 0 1 0
% 0 0 5 0

Pelvic girdle
pubic incomplete ossification or not ossified N 0 1 0 2
% 0 1 0 2
Litters N 0 1 0 2
% 0 5 0 12

Metacarpal or metatarsal
asymetric ossification N 1 2 0 0
% 1 2 0 0
Litters N 1 2 0 0
% 5 10 0 0
less than 3/3.5 ossified N 0 2 2 3
% 0 2 2 3
Litters N 0 2 2 3
% 0 10 9 18

FETAL MALFORMATIONS

Sternebra N 0 1 0 0
split % 0 1 0 0
Litters N 0 1 0 0
% 0 5 0 0
Ribs N 0 1 0 0
bifurcate % 0 1 0 0
Litters N 0 1 0 0
% 0 5 0 0
Thoraric centrum
dumb-bell shaped or bipartite (or bipartite asymmetric) and
cartilage dumb-bell shaped N 1 0 0 0
% 1 0 0 0
Litters N 1 0 0 0
% 5 0 0 0
Pectorale girdle N 1 0 0 0
scapula bent % 1 0 0 0
Litters N 1 0 0 0
% 5 0 0 0
Hindlimb N 0 1 0 0
femur, tibia, fibula short % 0 1 0 0
Litters N 0 1 0 0
% 0 5 0 0
fibula-calcanaeus, fibula-talus abnormal articulation N 0 0 0 1
% 0 0 0 1
Litters N 0 0 0 1
% 0 0 0 6

Remarks: * = p < 0.05; CH2
* = p < 0.05; CH2
** = p < 0.01; CH2
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of examined fetuses was 113, 109, 124 and 98 in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

Malformations:
One fetus was found with microphthalmy and one with situs inversus totalis in the control group. One fetus had multiple malformations (microphthalmy (bilateral); small kidney, small and malpositioned adrenal (unilateral) in the 100 mg/kg bw/day group. The right ovary was absent in this fetus and a technical error was not excluded. Considering that no other malformations were found in the test item treated groups at visceral examination this was judged to be likely unrelated from the treatment.

Variations:
Hydroureter and malpositioned testes (upper) was found with a very low incidence also in the control group and was judged to be unrelated from the treatment.


APPENDIX VII/A RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
(percentile litter means and SD)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000
VISCERAL EXAMINATION
Litters examined N 20 20 22 17
Fetuses examined N 113 109 124 98
Fetuses with abnormalities Mean 3,5 1,8 0 0,8
SD 7,48 5,67 0 3,46
Variation Mean 2 0,8 0 0,8
SD 6,29 3,73 0 3,46
Malformation Mean 1,5 1 0 0
SD 4,78 4,47 0 0


Remarks: * = p < 0.05
** = p < 0.01
U = Mann-Whitney U - test Versus Control
DN = Duncan's multiple range test


APPENDIX VII/A RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
(sum, %)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000
VISCERAL EXAMINATION
Litters examined N 20 20 22 17
Fetuses examined N 113 109 124 98
Fetuses with abnormalities N 4 2 0* 1
% 4 2 0 1
Litters N 4 2 0* 1
% 20 10 0 6
Variation N 2 1 0 1
% 2 1 0 1
Litters N 2 1 0 1
% 10 5 0 6
Malformation N 2 1 0 0
% 2 1 0 0
Litters N 2 1 0 0
% 10 5 0 0

Remarks:
* = p < 0.05; CH2
** = p < 0.01; CH2


APPENDIX X TYPES OF VISCERAL ABNORMALITIES
(sum, %)
DOSE GROUPS (mg/kg bw/day Control 100 300 1000

Number of Dams N 20 20 22 17
Fetuses examined N 113 109 124 98
Fetuses with abnormalities N 4 2 0* 1
% 4 2 0 1
Litters N 4 2 0* 1
% 20 10 0 6
Variation N 2 1 0 1
% 2 1 0 1
Litters N 2 1 0 1
% 10 5 0 6
Malformation N 2 1 0 0
% 2 1 0 0
Litters N 2 1 0 0
% 10 5 0 0

FETAL VARIATIONS

Ureters N 1 0 0 1
% 1 0 0 1
Litters N 1 0 0 1
% 5 0 0 6
Gonads N 1 1 0 0
% 1 1 0 0
Litters N 1 1 0 0
% 5 5 0 0


FETAL MALFORMATIONS

Eyes N 1 0 0 0
microphthalmy % 1 0 0 0
Litters N 1 0 0 0
% 5 0 0 0
General N 1 0 0 0
situs inversus totalis % 1 0 0 0
Litters N 1 0 0 0
% 5 0 0 0
Multiple malformed*** N 0 1 0 0
% 0 1 0 0
Litters N 0 1 0 0
% 0 5 0 0
Remarks:
* = p < 0.05; CH2 ***=microphthalmy (bilateral); kidney small, (right); adrenal small, half of left (right) and malpositioned, ovary absent (right) /ovary technical error is not excluded
** = p < 0.01; CH2
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
significantly increased growth retardation in fetuses at 1000 mg/kg bw (variation no dose response)
incomplete ossification in all dose groups (significantly increased at 300 mg/kg bw); no dose response

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related adverse effects observed at any dose level

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

Maternal effects

GROUPS (mg/kg bw/day):

Control

100

300

1000

NUMBER OF DAMS:

20

20

22

17

Corpora Lutea

13.6

13.2

13.3

13.6

Preimplantation Loss %

6.3

6.3

7.7

12.2

Implantation

12.7

12.3

12.2

11.9

Early Embryonic Death %

8.1

6.6

6.3

2.7

Late Embryonic Death %

1.1

2.5

1.2

0.0

Dead Fetuses %

0.0

0.4

0.0

0.5

Postimplantation Loss %

9.3

9.5

7.5

3.1*

Total Intrauterine Mortality %

15.0

15.3

14.4

15.3

Viable fetuses

11.6

11.1

11.3

11.5

Male fetuses %

56.5

46.6

47.1

53.1

Female fetuses % 

43.5

53.4

52.9

46.9

Fetal effects

EXTERNAL EXAMINATION

Control

100

300

1000

Litters examined 

20

20

22

17

Fetuses examined 

231

222

249

195

Fetuses with abnormalities

4.4

8.0

3.6

9.4

Variation

4.4

7.5

3.6

9.4

Malformation 

0.0

0.5

0.0

0.0

Retarded in body weight

4.4

8.0

3.6

9.4

VISCERAL EXAMINATION

 

 

 

 

Litters examined 

20

20

22

17

Fetuses examined

113

109

124

98

Fetuses with abnormalities

3.5

1.8

0.0

0.8

Variation 

2.0

0.8

0.0

0.8

Malformation

1.5

1.0

0.0

0.0

SKELETAL EXAMINATION

 

 

 

 

Litters examined

20

20

22

17

Fetuses examined

118

113

125

97

Fetuses with abnormalities

10.0

12.1

19.2

13.4

Variation

8.3

9.1

19.2

12.6

Malformation

1.7

2.9

0.0

0.8

 

Applicant's summary and conclusion

Conclusions:
Based on the results of the study no developmental toxicity was observed at 1000 mg/kg bw. Maternal effects (decreased food consumption (during day 5-14)) were observed at 300 mg/kg bw
Executive summary:

Twenty two sperm positive female rats were exposed orally to the substance from day 5 to 19 of gestation at 0, 100, 300 and 1000 mg/kg bw in a test according to OECD 414. Maternal effects were limited to a decreased food consumption during day 5 to 14 of the test period. No mortality, clinical signs and macroscopic changes were observed in the dams. Evaluations for developmental and fetal effects are based on 20, 20, 22 and 17 pregnancies. No effects on numbers of corpora lutea, implantations, resorptions, live fetuses, sex ratio and fetal weight were observed. No treatment related effects were found in external, visceral and skeletal examinations of the fetuses. The NOAEL for maternal toxicity is set at 1000 mg/kg bw (NOEL 300 mg/kg bw) and the NOAEL for developmental effects is 1000 mg/kg bw.