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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-11 to 2010-11-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
479-930-8
EC Name:
-
Cas Number:
613222-52-9
Molecular formula:
C40H76N2O4
IUPAC Name:
(3E)-3-({6-[(E)-[3-(dodecanoyloxy)-2,2-dimethylpropylidene]amino]hexyl}imino)-2,2-dimethylpropyl dodecanoate

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Hygienic level at arrival: SPF,
- Hygienic level during the study: Good conventional
- Number of animals: 48 males, 48 females, 12 animals/sex in the control and dose groups
- Sex: Males and nulliparous, non pregnant females
- Age of animals at start: Male animals: 10 – 13 weeks old, Female animals: 10 – 12 weeks old
- Body weight range at start: Male animals: 304 – 379 g, Female animals: 173 – 221 g, variation at start: < 20 % of mean group weight of each sex.
- Acclimatization time: 14 days
- Housing:
Before mating: 2 animals of the same sex/ cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: 2 animals / cage
- Diet: ssniff® SM R/M-Z+H produced by ssniff Spezialdiäten GmbH, D-59494 Soest

ENVIRONMENTAL CONDITIONS
- Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- Temperature: 22 ± 3 °C
- Relative humidity: 30 - 70 %
- Ventilation: 8-12 air exchanges/hour by central air-condition system.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Oleum helianthi (sunflower oil)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in sunflower oil, at concentrations of 30, 120 or 500 mg/mL. Formulations were prepared in the laboratory of ATRC either daily or for one or two days in advance on two occasions.

VEHICLE
- Justification for use and choice of vehicle: Sunflower oil ((Oleum helianthi)) was a suitable vehicle to facilitate formulation analysis for the test item. The suitability of the chosen vehicle for the test item (stability and homogeneity) was analytically proven.
- Concentration in vehicle: 30, 120 and 500 mg/mL
- Amount of vehicle: 2 mL/kg bw
- Lot/batch no.: 2010.05.13
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation occured
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability, concentration and homogenous distribution of test item in sunflower oil were confirmed analytically. Formulations were stable in sunflower oil at 25 mg/mL and 500 mg/mL concentration levels at least for 72 hours in a refrigerator (5 ± 3°C) and at least 6 hours at room temperature. Sika Härter LH concentrations in the dosing solutions varied in range of 87 % and 104 % in comparison to the nominal values.
Duration of treatment / exposure:
Males: 43 days (including one re-mating (4 days))
Females: 58 - 61 days (including one re-mating (4 days))
Frequency of treatment:
Once a day (7 days/week basis)
Details on study schedule:
Males:
Acclimatization period: 14 days
Pre-mating period: 14 days
Mating period: 14 days
Post-mating: 11 days

Females:
Acclimatization period: 14 days
Pre-mating period: 14 days
Mating period: 14 days
Gestation period: 22-23 days
Lactation period: 4-6 days
F1 offspring was terminated on postnatal days 4, 5 or 6. Non-pregnant females were necropsied 24, 25 or 27 days after sperm was observed in the vaginal smear.
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
240 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose setting is based on findings obtained in a previous oral toxicity study (Study No: 04/915-100P; test facility LAB International Research Centre Hungary Ltd.) with Sika Härter LH in rats and in agreement with the Sponsor.
- Rationale for animal assignment: All parental (P) male and female animals were sorted according to body weight and divided to weight groups aided by a computerized calculation. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomisation to ensure that the mean weight of animals from all test groups were as uniformly as practicable.
Positive control:
Not required.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations: signs of morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, prior to and during the mating until necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: first day of dosing (day 0), weekly thereafter and on the day of necropsy (male animals); first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 20 and on post-partal days 0 (within 24 hours after parturition) and 4, as well as on the day of necropsy (parent females); body weight of the female animals was weighed on gestational days 10 and 17

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule: weekly

OTHER:
- Examination of Placental Sign: All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on days 13 and 14 of the gestation period.
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.
Sperm parameters (parental animals):
The results of the determination of absolute and relative organ weights did not demonstrate any test item related organ weight alterations. There were no significant differences between the control and test item treated groups regarding the examined organ weights.

Parameters examined in P male parental generations:
testis weight, epididymis weight, other: weight of prostate and seminal vesicles with coagulating gland; histopathology of testes, epididymides, seminal vesicles
Litter observations:
STANDARDISATION OF LITTERS
- Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day when parturition was complete) with an accuracy of 0.01 g, and day 4 post-partum with an accuracy of 0.1 g.

PARAMETERS EXAMINED
The following parameters were examined in F1offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- All animals were sacrificed under Isofluran anesthesia by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [1] were prepared for microscopic examination and weighed, respectively
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed on postnatal days 4, 5 or 6.
- These animals were subjected to postmortem examinations macroscopic examination as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations
Statistics:
- The statistical evaluation of appropriate data (marked †above) were 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 access the significance of inter-group differences. Getting significant result 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 if feasible.
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"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs at any dose level (60, 240 and 1000 mg/kg bw/day). Alopecia was observed on the abdomen and hind limbs in two dams (no. 327 at 240 mg/kg bw – 1/12 - and no. 423 at 1000 mg/kg bw/day – 1/12) from gestational day 3 and 14, respectively, up to the termination. Wounds on these areas were also noted for animal no. 327 during the lactation period. Alopecia and wounds were considered to be individual signs, meaning that they may occur commonly also in untreated animals of this rat strain.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of male rats from all dose groups was similar to that of the control group animals throughout the entire treatment period. Statistically significant differences were noted for body weight gain data at 240 and 1000 mg/kg bw/day during the pre-mating period. The body weight gain at 240 mg/kg bw/day was slightly but significantly lower than in the control group between post-mating days 28 and 35 (App. 3.A, page 2 of 5). The summarized body weight gain (i.e. body weight gain between day 0 and termination) was also slightly lower compared to the value of the control group at 240 and 1000 mg/kg bw/day. There were no significant differences (statistical or biological) of body weight, body weight gain and total body weight gain data between all groups of female animals during the entire study (pre-mating, mating, gestation and lactation periods). In summary, there was no clear evidence of a test item related effect on the body weight development. Statistically significant differences in body weight gain of male animals at 240 and 1000 mg/kg bw/day did not show a clear dose related response and did not result in significant body weight differences comparing to their control value, therefore these were not considered toxicologically relevant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Compared to their appropriate control group, a slightly but statistically significantly lower mean daily food consumption was noted in male animals at 60, 240 and 1000 mg/kg bw/day during the first week of the pre-mating period and in male and female animals at 1000 mg/kg bw/day during the second week of the pre-mating period.
No further statistically significant differences occurred regarding the daily mean food consumption during all other phases of the present study.
In summary, statistical significances in food consumption were not considered to be toxicologically relevant as these occurred independently from doses (male animals during week 1 of the mating period) and were of a low degree (weeks 1 and 2 of mating period, male and female animals, respectively).
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
In the male animals the investigated, organs of the reproductive system (testes, epididymides, seminal vesicles, prostate, coagulative gland) and the pituitary gland were histologically normal in all groups - including those animals that did not mate or where pairing did not result in live offspring. The various spermatogonic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoon, and the interstitial cells were similar in quantity and morphologically in the testes of all animals investigated. Histologically, epididymides, seminal vesicles, prostate, coagulating gland and pituitary gland were normal in all cases as well.
In the female animals including not mated and non pregnant animals, the ovaries had a normal structure characteristic for the species, age and phase of sexual cycle. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals.
In single animals, dilatation of uterine horns (i.e. hydrometra; 1/12 control), pyelectasia (1/1 female at 60 mg/kg bw/day; 1/1 male at 240 mg/kg bw/day; 1/1 female at 1000 mg/kg bw/day) and slightly less than normal quantity of secretion in seminal vesicle on one side (male animal 1/12 in control group) were observed.
Pyelectasia in a slight degree occurred in a single treated animal without degenerative or inflammatory lesions or fibrosis. This is a common finding in rats and therefore has no pathological significance. Dilatation of uterine horns and less than normal quantity of secretion in seminal vesicles were noted for control animals therefore these had no toxicological significances in this study.
In summary, histological examination of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides, prostate, seminal vesicles with coagulating gland) and pituitary did not reveal any toxic or other test item related lesions at 60, 240 or 1000 mg/kg/bw/day doses.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
-There were no test item related clinical signs at any dose level (60, 240 and 1000 mg/kg bw/day).
Alopecia was observed on the abdomen and hind limbs in two dams at 240 mg/kg bw – 1/12 - and at 1000 mg/kg bw/day – 1/12) from gestational day 3 and 14, respectively, up to the termination. Wounds on these areas were also noted for animal no. 327 during the lactation period.
Alopecia and wounds were considered to be individual signs, meaning that they may occur commonly also in untreated animals of this rat strain.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
-The mean body weight of male rats from all dose groups was similar to that of the control group animals throughout the entire treatment period.
Statistically significant differences were noted for body weight gain data at 240 and 1000 mg/kg bw/day during the pre-mating period. The body weight gain at 240 mg/kg bw/day was slightly but significantly lower than in the control group between post-mating days 28 and 35 (App. 3.A, page 2 of 5). The summarized body weight gain (i.e. body weight gain between day 0 and termination) was also slightly lower compared to the value of the control group at 240 and 1000 mg/kg bw/day.
There were no significant differences (statistical or biological) of body weight, body weight gain and total body weight gain data between all groups of female animals during the entire study (pre-mating, mating, gestation and lactation periods).
In summary, there was no clear evidence of a test item related effect on the body weight development. Statistically significant differences in body weight gain of male animals at 240 and 1000 mg/kg bw/day did not show a clear dose related response and did not result in significant body weight differences comparing to their control value, therefore these were not considered toxicologically relevant.

FOOD CONSUMPTION
- Compared to their appropriate control group, a slightly but statistically significantly lower mean daily food consumption was noted in male animals at 60, 240 and 1000 mg/kg bw/day during the first week of the pre-mating period and in male and female animals at 1000 mg/kg bw/day during the second week of the pre-mating period.
No further statistically significant differences occurred regarding the daily mean food consumption during all other phases of the present study.
In summary, statistical significances in food consumption were not considered to be toxicologically relevant as these occurred independently from doses (male animals during week 1 of the mating period) and were of a low degree (weeks 1 and 2 of mating period, male and female animals, respectively).

DELIVERY DATA OF DAMS
- There were no differences between the control group and test item treated groups in the delivery data of dams.
There were no significant difference between the control and test item treated groups in percent and litter mean of corpora lutea, implantation sites and intrauterine mortality.
The percentage of dams delivered (calculated by the number of pregnant females), percentage of dams with live-borns and stillborns only, and the live birth index were similar in all groups. No test item related effect was found either in the litter means of number of total births, number of live-borns and stillborns. The mean of duration of pregnancy was similar to control in all test item treated groups.

REPRODUCTIVE PERFORMANCE
- No test item related effect was found on the reproductive ability of male and female animals at any dose level tested.
The number and percentage of mated and fertile male animals, as well as the copulatory and fertility indices were not affected by the treatment. The number and percentage of fertile males, copulatory and fertility indices were similar in the control and all test item treated groups.
There were no differences between the control group and test item treated groups in the number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestation indices. The number and percentage of non-pregnant and pregnant animals and number of pregnant animals with liveborns were also similar to the appropriate control values in all test item treated groups. The mean pre-coital interval was similar in the control group and the test item treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- The results of the determination of absolute and relative organ weights did not demonstrate any test item related organ weight alterations. There were no significant differences between the control and test item treated groups regarding the examined organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- In male animals, smaller than normal seminal vesicle (one-sided; 1/12, control) and pyelectasia (1/12 at 240 mg/kg bw/day) were observed.
In the female control group, an ovarian cyst (one-sided; 1/12) and hydrometra (1/12) were found in one animal each.
In the 60 mg/kg bw/day group, one-side pyelectasia was noted for one female animal (1/12).
In the 240 mg/kg bw/day group, one-side ovarian cyst (1/12), as well as alopecia and wounds on the abdomen (1/12) were detected in females.
In female animals of the 1000 mg/kg bw/day group, both sides pyelectasia (1/12) and ovarian cyst (1/12) and alopecia on the abdomen and hind limb (1/12) were observed.
In summary, no macroscopic alterations were found at the necropsy that were clearly test item related. Smaller than normal seminal vesicles were present in a single animal of the control group. Pyelectasia and alopecia are species-specific alterations, which commonly occur in animals of this species and strain. The ovarian cyst found were considered incidental since they occurred in single animals of the control group and test item groups (240 and 1000 mg/kg bw/day). Hydrometra due to the sexual cycle of female animals is a species-specific alteration. All observed effects can be seen occasionally in experimental rats and were not considered as test item related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- In the male animals the investigated, organs of the reproductive system (testes, epididymides, seminal vesicles, prostate, coagulative gland) and the pituitary gland were histologically normal in all groups - including those animals that did not mate or where pairing did not result in live offspring. The various spermatogonic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoon, and the interstitial cells were similar in quantity and morphologically in the testes of all animals investigated. Histologically, epididymides, seminal vesicles, prostate, coagulating gland and pituitary gland were normal in all cases as well.
In the female animals including not mated and non pregnant animals, the ovaries had a normal structure characteristic for the species, age and phase of sexual cycle. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals.
In single animals, dilatation of uterine horns (i.e. hydrometra; 1/12 control), pyelectasia (1/1 female at 60 mg/kg bw/day; 1/1 male at 240 mg/kg bw/day; 1/1 female at 1000 mg/kg bw/day) and slightly less than normal quantity of secretion in seminal vesicle on one side (male animal 1/12 in control group) were observed.
Pyelectasia in a slight degree occurred in a single treated animal without degenerative or inflammatory lesions or fibrosis. This is a common finding in rats and therefore has no pathological significance. Dilatation of uterine horns and less than normal quantity of secretion in seminal vesicles were noted for control animals therefore these had no toxicological significances in this study.
In summary, histological examination of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides, prostate, seminal vesicles with coagulating gland) and pituitary did not reveal any toxic or other test item related lesions at 60, 240 or 1000 mg/kg/bw/day doses.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
for reproductive performance
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean litter weight of the high dose group (1000 mg/kg bw/day) was statistically significantly lower (14 %) when compared to the control group on postnatal days 0 and 4. During that time period, the mean litter weight gain was also lower (14 %) compared to the control group.
However, there were no significant differences between the control and test item treated groups in the mean individual body weigh and body weight gain of pups.
In summary, the statistical significances found in the litter weight were of a minor degree and were within historical control ranges therefore they were considered to be of no toxicological relevance.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group, two pups were found dead and subjected to necropsy on postnatal day 0. In one of them, the performed lung flotation test was negative, indicating that this pup was stillborn. Autolysis of abdominal organs did not allow further inspection. Results of the lung flotation test of the second pup indicated that it died after delivery. No macroscopic alterations were found in the organs and tissues of the second pup.
In the 1000 mg/kg bw/day group, one pup was found dead on day 0. A lung flotation test performed was negative, indicating that this pup was stillborn. Macroscopic alterations were not found.
In summary, no test item related macroscopic alterations were found in offspring subjected to gross pathological examination. The number of stillborns was equal in the control group and high dose group.
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
-There was no extra uterine mortality in the test item treated groups. The only dead pup was found in the control group. The survival indices were similar between all groups.

CLINICAL SIGNS (OFFSPRING)
- There were no clinical signs in pups in the control group and in the test item treated groups.

BODY WEIGHT (OFFSPRING)
- The mean litter weight of the high dose group (1000 mg/kg bw/day) was statistically significantly lower (14 %) when compared to the control group on postnatal days 0 and 4. During that time period, the mean litter weight gain was also lower (14 %) compared to the control group.
However, there were no significant differences between the control and test item treated groups in the mean individual body weigh and body weight gain of pups.
In summary, the statistical significances found in the litter weight were of a minor degree and were within historical control ranges therefore they were considered to be of no toxicological relevance.
(Historical control values: Body weight: postnatal day 0: 63.33 g, SD: 19.45, n=15; postnatal day 04: 108.04 g, SD: 31.06, n=15; Body weight gain between postnatal days 0 and 4: 45.06 g, SD: 14.45, n=15.)

SEXUAL MATURATION (OFFSPRING)
- There were no differences between control and test item treated groups in the ratio or in the litter means of genders.

GROSS PATHOLOGY (OFFSPRING)
- In the control group, two pups were found dead and subjected to necropsy on postnatal day 0. In one of them, the performed lung flotation test was negative, indicating that this pup was stillborn. Autolysis of abdominal organs did not allow further inspection. Results of the lung flotation test of the second pup indicated that it died after delivery. No macroscopic alterations were found in the organs and tissues of the second pup.
In the 1000 mg/kg bw/day group, one pup was found dead on day 0. A lung flotation test performed was negative, indicating that this pup was stillborn. Macroscopic alterations were not found.
In summary, no test item related macroscopic alterations were found in offspring subjected to gross pathological examination. The number of stillborns was equal in the control group and high dose group.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for SIKA Hardener LH 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 F1 generation the NOAEL was 1000 mg/kg bw/day.
Executive summary:

The test item was assessed in a reproduction/developmental toxicity screening test according to OECD guideline 421 and draft OECD guidance document 43. The test item was administered orally (by gavage) to Hsd.Brl.Han: Wistar rats at repeated doses of 60, 240 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 sunflower oil at concentrations of 30, 120 or 500 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 87 % to 104 % of nominal).

For the present study, 12 animals/sex/group were used. All animals of the parent (P) generation received test item or control item prior to mating (14 days) and throughout 14 days mating (and in one case an attempted re-mating of four days, for details see chapter “Mating Procedure”). Test item or control item was administered to male animals until 11 days post mating. For females, test item was administered through the gestation period and up to lactation day 3, 4 or 5, i.e. up to the day before the necropsy. Observations included mortality, clinical symptoms, body weight, food consumption, mating, pregnancy and delivery process.

The dams were allowed to litter, and rear their young up to termination on days 4, 5 or 6 postpartum. All parental animals were subjected to gross pathology one day after the last treatment and offspring were euthanized. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups. In the low and middle dose groups, histopathological examinations were conducted on sexual organs of infertile males; in addition to females that did not exhibit signs of mating, did not deliver or were not pregnant as well as on the organs showing macroscopic findings at necropsy.

No mortality was observed in parent animals prior to scheduled necropsy. Clinical signs related to the test item were not detected during the entire treatment period. The general state and behaviour of animals was normal in all groups. The detected alopecia and minor wounds found in single animals at 240 mg/kg bw and at 1000 mg/kg bw/day were considered to be individual signs, meaning that they may occur commonly also in untreated animals of this rat strain.

The body weight development was undisturbed in each treatment group during pre-mating and post mating periods in male animals and in pre-mating, gestation and lactation periods in female animals. There were no test item related effects on food consumption of male and female animals at any dose level during the entire observation period.

There were no differences between the control and test item treated groups in the reproductive ability of male and female animals and in delivery data of dams.

Necropsy, organ weight and histopathological examinations of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) and pituitary did not reveal any toxic or other test item related lesions at any dose level.

There was no extra uterine mortality in the test item treated groups. The only dead pup was found in the control group. The survival indices were similar between all groups. There were no differences between control and test item treated groups in the ratio or in the litter means of genders. There were no clinical signs in pups in the control group and in the test item treated groups.

The mean litter weight of the high dose group (1000 mg/kg bw/day) was statistically significantly lower (14 %) when compared to the control group on postnatal days 0 and 4. During that time period, the mean litter weight gain was also lower (14 %) compared to the control group. However, there were no significant differences between the control and test item treated groups in the mean individual body weigh and body weight gain of pups. In summary, the statistical significances found in the litter weight were of a minor degree and were within historical control ranges therefore they were considered to be of no toxicological relevance.

In the control group, two pups were found dead and subjected to necropsy on postnatal day 0. In one of them, the performed lung flotation test was negative, indicating that this pup was stillborn. Autolysis of abdominal organs did not allow further inspection. Results of the lung flotation test of the second pup indicated that it died after delivery. No macroscopic alterations were found in the organs and tissues of the second pup.

In the 1000 mg/kg bw/day group, one pup was found dead on day 0. A lung flotation test performed was negative, indicating that this pup was stillborn. Macroscopic alterations were not found. In summary, no test item related macroscopic alterations were found in offspring subjected to gross pathological examination. The number of stillborns was equal in the control group and high dose group.

Under the conditions of the present study, SIKA Hardener LH caused no toxic alterations in male and female Hsd.Brl.Han: Wistar rats after repeated dose oral administration at 60, 240 or 1000 mg/kg bw/day. SIKA Hardener LH did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition). Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL for male rats: 1000 mg/kg bw/day

NOAEL for female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the male rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day