Dodecanoic acid, 1,1'-[1,6-hexanediylbis[nitrilo(2,2-dimethyl-1-propanyl-3-ylidene)]] ester

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

SIKA Hardener LH was tested in a reproduction/developmental toxicity screening test according to OECD guideline 421 and draft OECD guidance document 43. The no observed adverse effect level (NOAEL) for SIKA Hardener LH for parental effects was 1000 mg/kg bw/day (limit dose). 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.

Link to relevant study records

Reference

Reference 1

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

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

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.

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.

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.

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"

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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

Key result

Critical effects observed:

no

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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.

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Annex IX an extended one-generation study (OECD 443) should only be done, if the 28-day or 90-day studies indicate adverse effects on reproductive organs or tissues. An extended one-generation (OECD 443) study is not needed, as the available data from 28 d and 90 d repeated dose toxicity studies, reproduction/developmental screening study (OECD 421) and developmental toxicity study (OCED 414) do not indicate adverse effects on reproductive organs, fertility and development. The test item was administered by oral gavage to CRL:(WI)BR rats (n = 5/sex/dose) at dose levels of 0 mg/kg bw/day (vehicle only, control), 30 mg/kg bw/day, 160 mg/kg bw/day and 1000 mg/kg bw/day for 28 consecutive days. With regard to examinations on reproductive organs, gross pathology was performed on prostate, testes with epididymides, ovaries, uterus with vagina, seminal vesicles at the end of the treatment period and histological investigated. Testes and epididymides were weighed. Animals exhibited statistically significant decreased epididymides weights compared to the control group comparing absolute weights at 1000 mg/kg bw/d. However differences were not statistically significant on a relative basis. These differences in organ weight were in the range of historical control data and thus considered toxicologically insignificant. The dilatation of the uterine horns in all female groups, in connection with the hydrometra, is a neuro-hormonal phenomenon in connection with the sexual function of the inner genital organs. All histopathological differences were within the normal range of variation. Furthermore, an OECD 408 study was conducted to obtain information on possible health hazards likely arise from repeated exposure to the test item at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects. The test item was administered orally (by gavage) to Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 1000, 300 and 100 mg/kg bw/day corresponding to concentrations of 0, 500, 150 and 50 mg/mL, applied in a dose volume of 2 mL/kg bw for 90 or 91 days. Five animals/sex in the control and high dose groups assigned to the recovery groups were handled identically up to Day 89 and then observed without administration for another four weeks (recovery observations). Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at the end of the treatment period. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups, including recovery groups. In addition, the spleen was also processed histologically in all female animals of 300 mg/kg bw/day dose group based on macroscopic and histopathology findings in spleen at 1000 mg/kg bw/day to facilitate a better estimating of NOAEL. The results of study were interpreted comparing test item treated groups with respect to controls, which were administered concurrently with vehicle only. As a result of the study, a test item related influence on the estrous cycle was not detected (1000, 300 and 100 mg/kg bw/day). Sperm analysis did not reveal test item influence on the sperm cells (count, motility and morphology) at 1000 mg/kg bw/day. The aim of this reproduction/developmental toxicity screening test (OECD 421) was to provide initial information concerning the effect of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. The test item was administered orally (by gavage) once a day at 0 (vehicle control), 60, 240 or 1000 mg/kg/day doses and at concentrations of 30, 120 or 500 mg/mL corresponding to 2 mL/kg bw dose volume. 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). 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. The test item 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. The test item did not influence male and female reproductive performance (gonad function, mating behaviour, conception, pregnancy, parturition) and development of the F1 offspring from conception to day 4 (occasionally to days 5, 6) post-partum. Furthermore, a study using rats was conducted according to OECD Guideline 414 to examine the test item for its possible prenatal developmental toxicity. Groups of 22 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 24 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. The test item did not reveal any adverse effect on the pre- and post-implantation loss, number of implantation, sex distribution, body weight, and placental weight, external, visceral and skeletal development of the fetuses. Based on the above presented data no adverse effects on fertility are to be expected. Further testing would therefore most likely not lead to other results and would in conclusion not improve the hazard assessment of the substance. The available data are considered reliable and sufficient and therefore further testing (extended one-generation reproductive toxicity study) is not required and will not be carried out also taking animal welfare reasons into account.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP conform study according to guideline

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 421

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 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.

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

Effects on developmental toxicity

Description of key information

The test item was investigated in a developmental toxicity study according to OECD Guideline 414 in rats. 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 including teratogenicity): 1000 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-02 and 2016-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: 8.5 - 9.5 weeks (females), 34 - 36 weeks (males)
- Weight at study initiation: 162 - 206 g (females)
- Housing: Before mating: 1 - 3 females per cage 1 - 2 males per cage; Mating: 1 male and 1 - 3 females / cage; During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage
- Diet: ad libitum (ssniff® SM R/M-Z+H)
- Water: ad libitum (tap water)
- Acclimation period: 33 days for females, 180 days for males

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 43 - 62
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: Sunflower oil (Helianthii annui oleum raffinatum)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle (sunflower oil) in concentrations of 50 mg/mL, 150 mg/mL and 500 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility not longer than for three days and stored at 5 ± 3 °C until use.

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore Sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Amount of vehicle: 2 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing solutions (control of concentration) was performed in the Analytical Laboratory of Test Facility two times during the study. Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on 2 occasions. Similarly, five samples were taken from the vehicle and analyzed by a reverse phase HPLC method UV detection.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Recovery of Sika Hardener LH from sunflower oil was ≥ 97 % at ~25 and ~500 mg/mL concentrations, respectively.
A sufficient stability in the chosen vehicle was also verified over the range of relevant concentrations. The test item proved to be stable in the vehicle at ~25 and ~500 mg/mL concentration levels at least for 3 days in the refrigerator (5 ± 3 °C) and for 4 hours at room temperature. A separate analytical report – Toxi-Coop Study no. 644-100-0844– provided these data.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: one male: one to three females
- Length of cohabitation: two to four hours (until the number of sperm positive females per group achieved at least twenty two)
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Sperm positive females were treated from gestational day 5 to 19.

Frequency of treatment:

7 days/week

Duration of test:

Day 0 of gestation - Day 20 of gestation

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

80 males, 100 females to achieve at least 22 sperm positive females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting was based on findings obtained in a previous GLP 28-Day Oral Toxicity Study in the Rat with the test item (OECD 407, LAB Research Centre Hungary Ltd. 04/915-100P). The high dose was chosen with the aim of inducing toxic effects but no deaths or severe suffering. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

Maternal examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day (after treatment at approximately the same time)
- Behaviour and general conditions
- Signs of morbidity and mortality were made twice daily, at the beginning and end of the working period.

BODY WEIGHT: Yes
- Time schedule for examinations: at least once in the pre-mating period (female rats), on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (sperm positive females)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: 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

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus with cervix and ovaries

OTHER:
- Examination of placental signs (All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the 13th gestational day. If negative on day 13, the examination was repeated on day 14 of gestation.)

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
- Other: number of live and dead fetuses in each uterine horn

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Statistics:

The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result is significant Duncan’s Multiple Range test was used to access the significance of inter-group differences. If significance is 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.
Dams or litters were excluded from the data evaluation in cases of:
- Non pregnant females or dams (total exclusion)
Although these animals were excluded from the data evaluation the Study Report contains all data of these animals, too. A male/female fetus was considered as retarded in body weight, when its weight was below the average minus twofold standard deviation of the control male/female fetuses.

Historical control data:

Summary of Laboratory's historical control data is available.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed in the females.

Mortality:

no mortality observed

Description (incidence):

None of the females died in the course of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related significant differences in the body weight and body weight gain parameters of the dams in the test item treated groups versus control. The body weight gain was slightly but statistically significantly higher (p<0.01) in the 300 mg/kg bw/day group between gestation days 11 and 14 without a dose response and this difference was considered as incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no statistical difference in the food consumption of the dams among the experimental groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The gravid uterine weight which was measured in order to calculate the corrected body weight was slightly higher (p<0.01) in the 1000 mg/kg bw/day group, this was attributed to the slightly higher mean number of viable fetuses in this group and unrelated from the treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic alterations recorded for the dams during necropsy in the other experimental groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

The number of sperm positive females was 22 in all groups. There were two, five and three females each in the 100, 300 and 1000 mg/kg bw/day dose groups respectively with no implantation and no corpora lutea. These females were excluded from the evaluation. In total, on gestation day 20 there were 22, 20, 17 and 19 evaluated litters each in the control, 100, 300 and 1000 mg/kg bw/day groups respectively.

Other effects:

no effects observed

Details on maternal toxic effects:

INTRAUTERINE MORTALITY AND VIABLE FETUSES
There was no adverse effect indicated related to the administration in the mean percent of pre- and postimplantation loss (early embryonic, late- and fetal death), the mean number of implantations as well as in the mean number of viable fetuses in the test item treated groups. Moreover, the mean percent and whole number of pre- and postimplantation loss, early embryonic death and total intrauterine mortality were statistically significantly lower in the 1000 mg/kg bw/day group as well as the whole number of postimplantation loss in the 300 mg/kg bw/day group. In addition the mean number of viable fetuses was statistically significantly higher in the 300 and 1000 mg/kg bw/day group ((p<0.05 and 0.01 respectively).

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

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):

There was one malformed fetus found with agnathy in the control group during external examination.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The agnathy of the fetus in the control group was partially confirmed at skeletal examination (as a markedly hypoplastic mandible). Other malformations in this fetus of the skull (displaced tympanic rings, misshapen palate and sphenoid) as well as ribs (common articulation of two ribs to a vertebra) and vertebrae (Th IV bipartite, cartilage dumb-bell shaped; Th VII rotated, slight; Th VIII asymmetric dumb-bell shaped, cartilage and arches asymmetric; Th IX hemicentric, cartilage hemicentric; Th X displaced, cartilage asymmetric dumb-bell shaped; Th XII asymmetric bipartitewith bipartite cartilage were observed. A split and misshapen (wide) sternum was recorded for one fetus in the 1000 mg/kg bw/day group.
Considering the low incidence (one single fetus) and that split sternum occurs sporadically with low incidence according to the historical control data, this malformation was judged to be unrelated to an effect of the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no malformations found at visceral examination in the 300 and 1000 mg/kg bw/day groups. Internal hydrocephaly (dilated lateral ventricles) in one control fetus and situs inversus totalis in a fetus in the 100 mg/kg bw/day group were recorded.
Considering that situs inversus totalis occurs in fetuses with low incidence unrelated from the treatment according to the historical background data of Toxi-Coop Zrt. this malformation was judged to be unrelated from the treatment.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Please refer to "Details on embryotoxic/teratogenic effects"

Details on embryotoxic / teratogenic effects:

BODY WEIGHT OF FETUSES AND PLACENTAL WEIGHT
There were no treatment related differences in the body weight of the fetuses and placental weight. Statistically significantly lower placental weight was indicated for the fetuses (p<0.01) in the 300 and 1000 mg/kg bw/day dose group and in the relative placental weight in the 1000 mg/kg bw/day group. Considering that the values were within the historical control range, these differences were not attributed to an effect of the test item.

EXTERNAL EXAMINATION
The number of evaluated fetuses was 225, 218, 196 and 225 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
- Malformations: There was one malformed fetus found with agnathy in the control group during external examination.

- Variations: Body weight retardation (below 2.92 g for males and 2.68 g for females) was evaluated as an external variation. There were no treatment related differences among the experimental groups.

VISCERAL EXAMINATION
The number of evaluated fetuses was 225, 218, 196 and 225 in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
- Malformations: There were no malformations found at visceral examination in the 300 and 1000 mg/kg bw/day groups. Internal hydrocephaly (dilated lateral ventricles) in one control fetus and situs inversus totalis in a fetus in the 100 mg/kg bw/day group were recorded. Considering that situs inversus totalis occurs in fetuses with low incidence unrelated from the treatment according to the historical background data of Toxi-Coop Zrt. this malformation was judged to be unrelated from the treatment.

- Variations: Hydroureter was evaluated as variation and was found only in one fetus each in the control and 300 mg/kg bw/day group, hence there was no test item effect indicated.

SKELETAL EXAMINATION
The number of examined fetuses was 112, 110, 97 and 110 in the control, 100, 300 and 1000 mg/kg bw/day group respectively.
- Malformations: The agnathy of the fetus in the control group was partially confirmed at skeletal examination (as a markedly hypoplastic mandible). Other malformations in this fetus of the skull (displaced tympanic rings, misshapen palate and sphenoid) as well as ribs (common articulation of two ribs to a vertebra) and vertebrae (Th IV bipartite, cartilage dumb-bell shaped; Th VII rotated, slight; Th VIII asymmetric dumb-bell shaped, cartilage and arches asymmetric; Th IX hemicentric, cartilage hemicentric; Th X displaced, cartilage asymmetric dumb-bell shaped; Th XII asymmetric bipartitewith bipartite cartilage were observed. A split and misshapen (wide) sternum was recorded for one fetus in the 1000 mg/kg bw/day group.
Considering the low incidence (one single fetus) and that split sternum occurs sporadically with low incidence according to the historical control data, this malformation was judged to be unrelated to an effect of the test item.

- Variations: Incompletely or not ossified skull bones (including hyoid), incompletely ossified sternebra, sacral II vertebra or metacarpal/metatarsal, wavy ribs, bipartite sternebra, dumb-bell shaped, bipartite, asymmetrically ossified thoracic centra or slightly dumb-bell shaped cartilage as well as asymmetrically ossified metacarpal or metatarsal were evaluated as variations during the skeletal examination. There were no treatment related significant differences in the incidence of the different variations. The incidence of variations due to the markedly incompletely ossified skull bones was statistically significantly (p<0.01) and slightly higher in the 300 mg/kg bw/day group versus control. Considering the lack of dose response, this difference was judged to be unrelated from the treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

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 including teratogenicity): 1000 mg/kg bw/day

Executive summary:

The test item was examined for its possible prenatal developmental toxicity. Groups of 22 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 22 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The item in sunflower oil was stable at room temperature for four hours and for 3 days if stored in the refrigerator at the concentrations of 25 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 92 and 100 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

 

In total, on gestation day 20 there were 22, 20, 17 and 19 evaluated litters each in the control, 100, 300 and 1000 mg/kg bw/day groups respectively. None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed. There was no treatment related reduction indicated. Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. With regard to fetal examinations, there were no test item related differences in the fetal-and placental weight, body weight retardation and other external, visceral and skeletal variations. There were no malformed fetuses found in the experimental groups besides one control fetus with agnathy during external examination. There were no malformations found at visceral examination in the 300 and 1000 mg/kg bw/day groups. Internal hydrocephaly (dilated lateral ventricles) was found in one control fetus at visceral examination. Situs inversus totalis in a fetus in the 100 mg/kg bw/day group was considered to be unrelated from the treatment taking into accound the low incidence (one single fetus) and that situs inversus totalis occurs in fetuses with low incidence unrelated from the treatment according to the historical background data of Toxi-Coop Zrt. The same control fetus which had agnathy was found with multiple skeletal malformations (skull including mandible, ribs, and vertebrae). In the test item treated groups only a split and misshapen (wide) sternum was recorded for one fetus in the 1000 mg/kg bw/day group. Considering the low incidence and that split sternum occurs sporadically according to the historical control data, this malformation was judged to be unrelated to an effect of the test item.

 

In conclusion, oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test item at the dose levels of 100, 300 and 1000 mg/kg bw/day did not cause death, clinical signs and necropsy findings and did not influence the food intake and body weight development of the maternal animals. The test item did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation, sex distribution, body weight, and placental weight, external, visceral and skeletal development of the fetuses.

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 including teratogenicity): 1000 mg/kg bw/day

Reference 2

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to Annex IX, column 2 of REACH the decision on testing a second species in an OECD 414 study should be made based on the results obtained with the first species and also taking into account all other available data assessing the developmental toxicity of the substance to be registered. The test item was examined for its possible prenatal developmental toxicity according to OECD 414. Groups of 22 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 22 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The test item in sunflower oil was stable at room temperature for four hours and for 3 days if stored in the refrigerator at the concentrations of 25 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 92 and 100 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded. In total, on gestation day 20 there were 22, 20, 17 and 19 evaluated litters each in the control, 100, 300 and 1000 mg/kg bw/day groups respectively. None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed. No treatment related reduction in body weight and food consumption was observed. Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. With regard to fetal examinations, there were no test item related differences in the fetal-and placental weight, body weight retardation and other external, visceral and skeletal variations. There were no malformed fetuses found in the experimental groups besides one control fetus with agnathy during external examination. There were no malformations found at visceral examination in the 300 and 1000 mg/kg bw/day groups. Internal hydrocephaly (dilated lateral ventricles) was found in one control fetus at visceral examination. Situs inversus total is in a fetus in the 100 mg/kg bw/day group was considered to be unrelated from the treatment taking into account the low incidence (one single fetus) and that situs inversus totalis occurs in fetuses with low incidence unrelated from the treatment according to the historical background data of Toxi-Coop Zrt. The same control fetus which had agnathy was found with multiple skeletal malformations (skull including mandible, ribs, vertebrae). In the test item treated groups only a split and misshapen (wide) sternum was recorded for one fetus in the 1000 mg/kg bw/day group. Considering the low incidence and that split sternum occurs sporadically according to the historical control data, this malformation was judged to be unrelated to an effect of the test item. The test item did not reveal any adverse effect on the pre- and post-implantation loss, number of implantation, sex distribution, body weight, placental weight, external, visceral and skeletal development of the fetuses in this OECD 414 study. Furthermore, a reproduction/developmental toxicity screening test according to OECD 421 was performed to provide initial information concerning the effect of the test item on male and female reproductive performance. 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 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. 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). 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. As a result of this OECD 421 study, the test item did not influence male and female reproductive performance (gonad function, mating behaviour, conception, pregnancy, parturition). Based on these results, it is not expected that further testing in a second species would lead to other results and would improve the hazard assessment of the substance. The available studies were considered reliable and sufficient for assessment of the developmental toxicity and, besides taking animal welfare reasons into account, there is no need to confirm the obtained results in a further study.

Species:

rabbit

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP conform study according to guideline

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 414

The test item was examined for its possible prenatal developmental toxicity. Groups of 22 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 100, 300 and 1000 mg/kg bw/day from day 5 up to and including day 19 post coitum. A control group of 22 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 2 mL/kg bw.

A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. The item in sunflower oil was stable at room temperature for four hours and for 3 days if stored in the refrigerator at the concentrations of 25 and 500 mg/mL. Analytical control of dosing solutions was performed on the first and last week of treatment. Concentrations of the test item in the dosing formulations varied in the acceptable range between 92 and 100 % of nominal concentrations at both analytical occasions confirming proper dosing.

During the study, mortality was checked and clinical observations were performed. Body weight and food consumption of the dams were also recorded. The day, when sperm was detected in the vaginal smear, was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.

After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

 

In total, on gestation day 20 there were 22, 20, 17 and 19 evaluated litters each in the control, 100, 300 and 1000 mg/kg bw/day groups respectively. None of the female died before scheduled necropsy and there were no clinical signs recorded. No treatment related necropsy findings were observed. There was no treatment related reduction indicated. Number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. With regard to fetal examinations, there were no test item related differences in the fetal-and placental weight, body weight retardation and other external, visceral and skeletal variations. There were no malformed fetuses found in the experimental groups besides one control fetus with agnathy during external examination. There were no malformations found at visceral examination in the 300 and 1000 mg/kg bw/day groups. Internal hydrocephaly (dilated lateral ventricles) was found in one control fetus at visceral examination. Situs inversus totalis in a fetus in the 100 mg/kg bw/day group was considered to be unrelated from the treatment taking into accound the low incidence (one single fetus) and that situs inversus totalis occurs in fetuses with low incidence unrelated from the treatment according to the historical background data of Toxi-Coop Zrt. The same control fetus which had agnathy was found with multiple skeletal malformations (skull including mandible, ribs, and vertebrae). In the test item treated groups only a split and misshapen (wide) sternum was recorded for one fetus in the 1000 mg/kg bw/day group. Considering the low incidence and that split sternum occurs sporadically according to the historical control data, this malformation was judged to be unrelated to an effect of the test item.

 

In conclusion, oral treatment of pregnant Hsd. Han: WISTAR rats from gestation day 5 up to day 19 (the day before Caesarean section) with the test item at the dose levels of 100, 300 and 1000 mg/kg bw/day did not cause death, clinical signs and necropsy findings and did not influence the food intake and body weight development of the maternal animals. The test item did not reveal any adverse effect on the pre- and postimplantation loss, number of implantation, sex distribution, body weight, and placental weight, external, visceral and skeletal development of the fetuses.

 

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 including teratogenicity): 1000 mg/kg bw/day

Justification for classification or non-classification

Based on the test results, no classification and labelling for toxicity to reproduction is required according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.