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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-04-07 until 2009-05-29
Reliability:
1 (reliable without restriction)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
and draft OECD guidance document 43
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Aldimine 1
- Physical state: Liquid
- Analytical purity: 96.1% (Aldimine group contents via acid titration)
- Lot/batch No.: UB2.398A/07
- Expiration date of the lot/batch: 2011-08-04
- Storage condition of test material: Keep tightly closed. Stored at room temperature at 20 ± 5° C in a dry place. Keep away from heat and open flame.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd., 1103 Budapest, Cserkesz u. 90

- Age at study initiation: Young adult rats, at least 10 weeks old at starting and 12 weeks at mating. The age range within the study was kept to the minimum practicable

- Weight at study initiation: Males: 337 - 416 g; females: 230-295 g

- Housing: Before mating: 4 animals of the same sex / cage. Mating: 1 male and 1 female / cage. Pregnant females were housed individually; the bedding material was suitable for nesting.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption from 500 mL bottle, ad libitum.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 23.7 °C
- Humidity (%): 35 - 69 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m


IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
- Justification for use and choice of vehicle (if other than water): As this test item is not soluble in water, PEG400 was considered the suitable vehicle to use for the preparation of dose formulations for oral administration, as instructed by the Sponsor and according to the analytical method
- Concentration in vehicle: The test item was formulated in PEG400 at concentrations of 8.75, 87.5 and 250 mg/mL . A constant treatment volume of 4 mL dose formulation/kg bw was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals determined at least weekly. In the first week of the pre-mating period, animals received the volumes based on the actual body weight on day 0.
- Lot/batch no. (if required): 1421464
Details on mating procedure:
- M/F ratio per cage: 1 male and 1 female / cage
- Length of cohabitation: Up to 5 days of pairing
- Proof of pregnancy: By vaginal plug and/or sperm in vaginal smear. The presence of the vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421, or gestation day GD0).
- After successful mating each pregnant female was caged (how): Pregnant females were housed individually; the bedding material was suitable for nesting.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of dose formulations (concentration and homogeneity) were conducted during the first and last week of treatment of the study from dose formulations administered to animals when all the concentrations were employed, depending upon the mating/delivery schedule. Dose formulations were homogenous. No SIKA Hardener LI was detected in the control samples; in the test item dose formulations. The measured concentrations ranged from 90.1 to 105% of nominal concentrations (8.75, 87.5, and 250 mg/mL). These results were considered suitable for the study purposes.
Duration of treatment / exposure:
The test item was administered daily by oral gavage, at similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology. Dosing of both sexes began after an appropriate acclimatisation (A) period and two weeks before mating and was continued up to and including the day before necropsy. Mating began soon after the animals attained full sexual maturity. Dosing was continued in both sexes during the mating period.
Males were dosed for 28 days, 14 days pre-mating (PM) and 14 days mating/post- mating period (M), then they were euthanised and subjected to necropsy examination.
Females were dosed for 14 days pre-mating (PM), for up to 14 days mating period (M), through gestation (up to 23 days) (G) and day 4 post-partum (PP/PN) with necropsy the following day (up to treatment day 52). The day of birth (viz. when parturition was complete) was defined as day 0 post-partum
Frequency of treatment:
Once a day
Details on study schedule:
Males:
Acclimation period: 13 days
Pre-mating period: 14 days (dosed period)
Mating/Post mating: 14 days (dosed period)
At the end of the mating/post mating period (after 28 days of dosing) the males were euthanised and subjected to necropsy examination.
Females:
Acclimation period: 13 days
Pre-mating period: 14 days (dosed period)
Mating period: Up to 14 days (dosed period)
Gestation period: Up to 23 days (dosed period)
After delivery lactation period of 4 days (dosed period). After the lactation period (day 5 post-partum) necropsy.
All F1 offspring were terminated on day 4 post partum.
The day of birth was defined as day 0 post-partum
Doses / concentrations
Remarks:
Doses / Concentrations:
35, 350, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected by the Sponsor based on a previous study conducted with this test item in rats
- Rationale for animal assignment (if not random): All parental (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomisation to ensure that animals of all test groups were as nearly as practicable of uniform weight. The grouping was controlled by SPSS/PC software, according to the actual body weight and with verification of the homogeneity/variability between/within the groups and cages.
Positive control:
No positive control was used

Examinations

Parental animals: Observations and examinations:
- Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
- Clinical observations for signs of ill health or reaction to treatment were made at least once daily, with detailed examination performed weekly.
- Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partal periods, and relevant parameters and/or indices were measured and/or calculated (female and male mating index, female and male fertility index, gestation index)
- Body weight and food consumption were measured at least weekly.
- Gross necropsy was conducted at the end of the treatment period. The absolute and relative organ weights of selected organs and tissues were determined. A histopathological examination was performed on the selected preserved organs and tissues of the animals of the control and high dose groups, and on abnormal tissues from low and mid dose groups.

The observations and examinations performed in parental males are detailed in the following:
- Clinical observations
- Body weight
- Body weight gain
- Food consumption
- Number of pairings
- Number of fertile pairings
- Number of infertile males
- Male mating index
- Male fertility index
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights)
- Histopathology findings

The observations and examinations performed in parental females are detailed in the following:
- Clinical observations
- Body weight
- Body weight gain
- Food consumption
- Number of pairings
- Number of pregnant females
- Number of sperm positive, but non-pregnant females
- Number of non mated females
- Female mating index
- Female fertility index
- Gestation index
- Duration of pregnancy (days)
- Number of corpora lutea / dams
- Number of implantations / dams
- Number of dams with live pups days 0 and 4
- Pre-implantation mortality
- Intrauterine mortality
- Total mortality (intra and extra uterine mortality)
- Necropsy findings
- Histopathology findings
Oestrous cyclicity (parental animals):
Females were already dosed 2 weeks before mating (a pre-mating period) with the objective of covering at least two complete oestrous cycles. During the mating period (up to 14 days), each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope, the presence of the vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421, or gestation day GD0). Results contributed to the calculations of female(and male) mating index and female (andmale) fertility index.
Sperm parameters (parental animals):
Parameters examined in male parental generation (sperm parameters):
The testes, epididymides (total and cauda), seminal vesicles with coagulating glands, prostate were weighed.
Microscopic observations regarding sperm parameters included:
In the testes observation of the following indications: Retention, spermatid, stage IX-XII, artrophy, tubular
In the epididymides observation of the following indications: Apoptosis, epithelial, cell infiltrate
In the prostate observation of the following indications: Inflammation, cell debris, cell infiltrate.
Litter observations:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that were significantly smaller than normal pups), and the presence of gross abnormalities, if any. Live pups were counted, sexed, weighed individually with an accuracy of 0.1 g within 24 hours of parturition.
All the litters were checked and recorded daily for the number of viable and dead pups.
Behaviour of the offspring was evaluated (in addition the behaviour of the dams towards the new-borns was observed: nesting behaviour (whether they made a nest from the bedding material and cover their new-borns or not). The efficiency of the suckling was observed by the presence of milk in the pups' stomach).
in detail:
- Mean pup body weight (per pup and per litter) on postnatal days 0 and 4
- Mean pup body weight gain (per litter) between postnatal days 0 - 4
- Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
- Survival index of pups on postnatal days 0 and 4
- Sex ratio % (on postnatal days 0 and 4)

Postmortem examinations (parental animals):
Gross necropsy was performed on each animal irrespective of the date of its death. One day after the last treatment, animals were sacrificed under pentobarbital anaesthesia. After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. Any abnormality was recorded with details of the location, colour, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The testes, epididymides (total and cauda), seminal vesicles with coagulating glands, prostate, and the female reproductive organs including ovary, uterus (with and without cervix), vagina, and brain and pituitary of all adult animals were weighed. Paired organs were weighed separately.
The weighed organs and all organs showing macroscopic lesions, of all adult animals were preserved (Bouin´s fixative)
Pups euthanized at day 4 postpartum were carefully examined at least externally for gross abnormalities.
Postmortem examinations (offspring):
Pups, killed at day 4 post-partum were only examined externally for gross abnormalities (in accordance with the OECD 421 Guideline).
Statistics:
The statistical evaluation of appropriate data was performed with the statistical 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 Multiple Range test was used to assess the significance of inter-group differences. Where significant result was obtained at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as required.
Reproductive indices:
The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index.
The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"
Offspring viability indices:
The following pup mortality and sex ration indices were calculated: Survival index, pre - implantation mortality, intrauterine mortality , total mortality, sex ratio. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

There were no significant differences between the control and test item treated groups with regard to reproductive ability or in the mating, fertility or gestation indices.
- Test item administration did not impact the duration of the mating period
- Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 days of pairing (cohabitation), with the exception of control female 1510 and high dose female 4509, for which mating period was prolonged for reasons not related to test item administration- No test item effect on the duration of pregnancy was observed. The females generally littered in 22 to 23 days.
- All the parturitions were normal
- There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition or post-partal period noted in the treated groups at dose levels up to and including 1000 mg/kg bw/day compared to control animals. There were no test item-related alterations in the delivery data of SIKA Hardener LI-treated dams as compared to the control value
- The number of corpora lutea were similar in the treated groups up to and including 1000 mg/kg bw/day, compared to females in the control groups
- The mean number of implantation sites appeared to be lower than control in the test item treated groups, with the lowest value in the high dose (12.75 vs. 16.18), likely due to females 4506 and 4507 (with 4, and 3 implantations, respectively). However, the differences did not attain statistical significance, the mean values were comparable with the mean historical control value (12.55, within a range from 2 to 18, Appendix 11) and these variations were not considered toxicologically significant in correlation with the treatment in the conditions of this study
- Total mortality (%) mean value was apparently higher than control in the low and high dose SIKA Hardener LI-treated groups, without attaining statistical significance and with no dose response in the mid dose group. In the low dose group, this was likely due to a relatively high intrauterine mortality in the female 2502. In the high dose group, the high intrauterine mortality was due to a high pre-implantation mortality in 2 females (4506, with 14 corpora lutea, 4 implantations and 4 live born pups, and 4507, with 13 corpora lutea, 3 implantations and no live born pup). In all the other females the preimplantation, intrauterine and total mortality values were low, within the historical control range and similar in the treated and control groups, thus, these changes were considered incidental and not regarded as an effect of the treatment
- Administration of SIKA Hardener LI at all dose levels examined was not associated with any test item-related macroscopic or microscopic findings in the parent animals surviving to scheduled termination. There were no treatment-related organ weight changes.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for parental effects
Effect level:
1 000 mg/kg bw/day
Sex:
male/female
Dose descriptor:
NOAEL
Remarks:
for reproduction parameters
Effect level:
1 000 mg/kg bw/day
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

- The postnatal mortality (%) mean values were low, showed minor differences with no dose response between the test item treated groups and were neither considered toxicologically significant, nor related to test item administration.
- Litter examination did not reveal any clinical, or macroscopic test item-related effects compared to observations noted in the control group. SIKA Hardener LI- administration had no adverse effect on the mean or total number of pups, or pup survival index. The sex ratio was similar in the control and treated groups and no significant variations were observed on PN4 vs. PN0.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
for pups growth and adverse effects
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

No remarks

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for SIKA Hardener LI for parental effects was 1000 mg/kg bw/day. For reproduction parameters, no effects were noted at any dose level, resulting in a NOAEL of 1000 mg/kg bw/day. For pup growth rate and adverse effects, the NOAEL was 1000 mg/kg bw/day.
Executive summary:

SIKA Hardener LI was assessed in a reproduction/developmental toxicity screening test according to OECD guideline 421. SIKA Hardener LI was administered orally (by gavage) to CRL:(WI)BR rats at repeated doses of 35, 350 and 1000 mg/kg bw/day compared to control animals, for 28 days in the male animals and up to 52 days in female animals. The dose setting was based on findings obtained in previous studies (see section 7.2 and 7.5). The test item was formulated in PEG400 at concentrations of 8.75, 87.5 and 250 mg/mL, corresponding to a 2 mL/kg bw dose volume. Analysis of dose formulations (concentration and homogeneity) were conducted during the first and last week of treatment of the study from all the concentrations employed. Recovery showed that dose formulations were homogenous and concentrations within an acceptable range of 100 ± 10 % (actual range 105-93% of nominal).

Repeated administration of SIKA Hardener LI did not result in any treatment related clinical effects. No adverse or toxicologically significant effects were noted and mean body weight, body weight gain and food consumption in treated groups compared to control animals.

There were no significant differences between control and test item treated groups for reproductive ability or in mating, fertility or gestation indices and duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 5 days of pairing (cohabitation). No test item effect on the duration of pregnancy was observed. The females generally littered in 22 to 23 days. All the parturitions were normal. There were no adverse effects, or biologically significant variations of the parameters related to pregnancy, parturition or post-partal period noted in the treated groups at dose levels up to and including 1000 mg/kg bw/day compared to control animals. There were no test item-related alterations in the delivery data of treated dams compared to controls. The number of corpora lutea was similar in the treated groups compared to controls. The mean number of implantation sites appeared to be lower in treated groups, but this observation had no statistical significance and was within historical controls, thus, not considered toxicologically significant. Total mortality (%) mean value was apparently higher than control in the low and high dose treated groups, without attaining statistical significance and with no dose response. In the low dose group, this was likely due to a relatively high intrauterine mortality in one of the female animals. In the high dose group, the high intrauterine mortality was due to a high pre-implantation mortality in 2 females. In all the other females the preimplantation, intrauterine and total mortality values were low, within the historical control range and similar in the treated and control groups, thus, these changes were considered incidental and not regarded as an effect of the treatment. No test item-related macroscopic or microscopic findings were observed and no treatment-related organ weight changes.

The postnatal mortality (%) mean values were low, showed minor differences with no dose response between the test item treated groups and were neither considered toxicologically significant, nor related to test item administration. There were no abnormalities in pups that were ascribed to the treatment: Litter examination did not reveal any clinical, or macroscopic test item-related effects compared to observations noted in the control group. SIKA Hardener LI administration had no adverse effect on the mean or total number of pups, or pup survival index. The sex ratio was similar in the control and treated groups and no external gross abnormalities were observed.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for SIKA Hardener LI for parental effects was 1000 mg/kg bw/day. For reproduction parameters, no effects were noted at any dose level, resulting in a NOAEL of 1000 mg/kg bw/day. For pup growth rate and adverse effects the NOAEL was 1000 mg/kg bw/day.