Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-09-18 to 2013-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD Guideline study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
other: Hsd.Brl.Han: of Wistar origin
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: females: 11.5-12 weeks, males: 12-13 weeks
- Weight at study initiation: 181-258 g
- Housing: Before mating: 3 females per cage 2 males per cage; mating: 1 male and 1-3 females / cage; during gestation: 1-2 sperm positive females per cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance", ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water: tap water ad libitum
- Acclimation period: 20 days for females and for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22 °C
- Humidity (%): 33 - 59 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore PEG 400 was used for preparing formulations appropriate for oral administration and according to the analytical method. PEG 400 is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: The test item was formulated in the vehicle in concentrations of 50 mg/mL, 100 mg/mL and 250 mg/mL.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control (concentration, homogeneity) of dosing solutions was performed in the Laboratory of Toxi-Coop Zrt. on the first and last weeks of treatment. The test item was analyzed using reverse phase HPLC method with UV detection.
Details on mating procedure:
- Impregnation procedure: The females were paired to males until the number of sperm positive females per group achieved at least twenty four.
- M/F ratio per cage: 1 male/1-3 females
- Length of cohabitation: 2-4 hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
The sperm positive females were treated from gestational day 5 to 19. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology. Control animals were handled in an identical manner to the test groups, except for treatment with the test item.
Frequency of treatment:
The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis every day at similar time.
Duration of test:
15 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 400 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses have been chosen by the Sponsor on the basis of a previous study (GLP OECD 421 Reproduction/Developmental Toxicity Screening Test of Aldimine 1 (CAS: 932742-30-8, identical with SIKA Hardener LI) in the Wistar Rat (Study code: 08/732-209P).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day, when signs of toxicity observed, animals were observed more frequently

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the female rats was measured once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured between gestation days 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 (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: the uterus with cervix and the left ovary were weighed, the number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted.

OTHER: Fetuses were removed from the opened uterus and assessed for viability.
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 (those subjected to visceral examination)
Statistics:
Data were individually recorded on data sheets, transferred, and compiled by computer or compiled manually. The statistical evaluation of data was performed with the program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance was the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Indices:
- Number of corpora lutea
- Number of implantations
- Number and percent of live fetuses
- Pre-implantation loss
- Post-implantation loss
- Sex distribution
- Fetal body weight (by sex)
- Fetal body weight (with sexes combined)
- External abnormalities/litter
- Visceral abnormalities/litter
- Skeletal abnormalities/litter

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
CLINICAL SIGNS
None of the females died or was in moribund state in the course of the study.
Salivation was recorded for 16 of 22 dams directly after treatment ca. 5 to 10 minutes long with a frequency of one to five days (between gestation days 15 and 19) in the 1000 mg/kg bw/day dose group. Since this was probably a consequence of the taste of the test item formulation, it was considered to be non adverse.
There were no clinical signs recorded for the dams in the 200 and 400 mg/kg bw/day dose groups.

BODY WEIGHT
There were no significant differences in the body weight of the dams in the experimental groups compared to control group.
A slight but statistically significant increase of body weight gain was indicated in the 400 and 1000 mg/kg bw/day groups between gestation days 8 to 11. The corrected body weight was statistically significantly higher in the 200, 400 and 1000 mg/kg bw/day groups than in the control. There was no dose response in the changes of these parameters.
The increase in the body weight and corrected body weight gain was judged to be incidental and not related to the administration of the test item to the dams.

INTRAUTERINE MORTALITY OF THE CONCEPTUSES, NUMBER OF IMPLANTIONS
There was no effect related to the administration of SIKA Hardener LI in the number of corpora lutea, preimplantation loss, intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution.
A lower preimplantation loss in the 400 mg/kg bw/day group was without a biological relevance

Effect levels (maternal animals)

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

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
EXTERNAL, VISCERAL AND SKELETAL EXAMINATION
The percentage of the litters with malformed fetuses was 15, 9, 10 and 9% in the control, 200, 400 and 1000 mg/kg bw/day groups, respectively.
The number of evaluated fetuses was 217, 235, 226 and 224 in the control, 200, 400 and 1000 mg/kg bw/day groups, respectively.
Malformations
At external examination there was one malformed fetus in the 1000 mg/kg bw/day dose group (0407138/9) with short mandible and maxilla, slightly protruding tongue and neck edema. This fetus was found to be malformed also at skeletal evaluation.
According to the experience with this species in this laboratory, similar findings occur sporadically without a relationship to the treatment, moreover, the historical control data contains agnathy as a more severe malformation than short mandible, thus the malformation of this fetus was judged to be incidental.
One fetus with microphtalmy and one with a retroesophageal origin of the right subclavian artery each in the 1000 mg/kg bw/day group was found at visceral examination. One fetus had a marked diaphragmatic hernia in the control group. Microphthalmy occurs sporadically also in control fetuses according to the historical control data of this laboratory. Both microphthalmy and retroesophageal origin of the right subclavian artery were found each in one single case, thus considered to be incidental and not attributed to the administration of the test item to the dams.
The incidence and percentage of skeletal malformation was equal in all groups (2%).
Variations
The incidence of external variations i.e. growth retarded fetuses was at the level of the control group. No effect due to the treatment was indicated. The visceral variations (moderately dilated lateral and IIIrd brain ventricles and a bilateral hydroureter) were found each in one fetus in the 400 mg/kg bw/day and 1000 mg/kg bw/day groups respectively. The incidence of these variations was below the historical control level, thus considered to be without a relationship to the treatment.
Incomplete or markedly incomplete ossification of the skull, incompletely ossified, bipartite or misaligned sternebrae (recorded with a percentage of 0-3%), wavy ribs, dumb-bell shaped or bipartite vertebral centra asymmetric ossification of metacarpal or metatarsal (found with a percentage of 0-5% and without any dose response) were evaluated as variations during the skeletal examination.
There was a slight increase without a statistical significance in the incidence of fetuses with skeletal variations in the 200, 400 and 1000 mg/kg bw/day groups and in the incidence of fetuses with skeletal abnormalities (both within the historical control level and at a similar level for all of these groups) though that the different type of the individual variations was with a very low incidence. These differences were judged to be due to the low current control values (below the historical control level) and were not attributed to an effect of the test item.

INRAUTERINE MORTALITY, VIABLE FETUSES, SEX DISTRIBUTION
There was no effect related to the administration of SIKA Hardener LI in the number of corpora lutea, preimplantation loss, intrauterine mortality of the conceptuses, the number of implantations, viable fetuses and their sex distribution.
A lower preimplantation loss in the 400 mg/kg bw/day group was without a biological relevance.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, under the test conditions chosen the test item SIKA Hardener LI did not cause any adverse effects in pregnant rats or in the fetuses.
Executive summary:

In a study conducted according to OECD Guideline 414, groups of of 24 sperm-positive female Hsd. Brl. Han: Wistar rats were treated with SIKA Hardener LI by oral administration (gavage) daily at three dose levels of 0, 200, 400 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. The treatment volume was 4 mL/kg b wand PEG 400 was used as vehicle. The test item did not cause death, adverse clinical signs and necropsy findings in the dose groups. SIKA Hardener LI did not reveal any adverse effect on the pregnancy, body weight, corrected body weight, and food consumption data of the dams, on the gravid uterine weight, the intrauterine mortality of the conceptuses, the number of viable fetuses and their sex distribution, the fetal and placental weight. SIKA Hardener LI did not increase the incidence of external and visceral and skeletal variations and caused no external, visceral or skeletal malformations in the fetuses. Based on these observations the No Observed Effect Level (NOEL) was determined as follows:

NOEL maternal toxicity: 400 mg/kg bw/day

NOEL developmental toxicity: 1000 mg/kg bw/day

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL maternal toxicity: 1000 mg/kg bw/day

NOAEL developmental toxicity: 1000 mg/kg bw/day