Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an OECD Test Guideline 421 reproduction and developmental toxicity screening study, to GLP, parental rats (12/sex/group) were administered tetraamminepalladium dichloride by gavage at 0 (in aqueous ammonium chloride), 4, 20 or 100 mg/kg bw/day. No adverse effects on the dams, reproductive parameters, or on development of offspring, were observed at any dose, resulting in a reproductive toxicity NOAEL of 100 mg/kg bw/day (Török-Bathó, 2015).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
24 June 2014 - 09 August 2014 (last necropsy)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, conducted to GLP, on closely-related surrogate
Justification for type of information:
Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the diacetate, dichloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide and dichloride are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult rats, at least 10 weeks old at starting and at least 12 weeks at mating.
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no data
- Housing: Type II and III polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany offered ad libitum
- Water (e.g. ad libitum): tap water from municipal supply as for human consumption from 500 ml bottle ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-23.8 °C (target range 22±3°C)
- Humidity (%): 40 - 70 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
Start of experiment (start of treatment): 24 June 2014
End of treatment: 22 July 2014 (males); 08 August 2014 (females)
End of experiment: 09 August 2014 (last necropsy)
Route of administration:
oral: gavage
Vehicle:
other: an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was used as the dosing vehicle.

The buffer was prepared as follows:
2.68 g ammonium chloride (Batch/Lot No.: BCBM5575V, Supplier: Sigma-Aldrich, Expiry: March 2015) was dissolved in 1000 ml distilled water (Batch/Lot No.: 0271113, Supplier: TEVA Pharmaceutical Works PLtd., Expiry: November 2016).
The pH was adjusted to 9 by adding approximately 1650-1850 µL of 29.1% ammonium hydroxide (Batch/Lot No.: SZBD2260V, Supplier: Sigma-Aldrich, Expiry: August 2015).

The first preparation was used for 7 consecutive days and was kept at room temperature. The next preparations were used for periods of up to 14 days, following confirmation of the stability, and the stock solution was kept refrigerated (2-8ºC). Dose solutions for treatment were diluted from the stock solution with aqueous ammonium-chloride buffer to the concentrations of 4.0 and 0.8 mg/mL, daily just before the treatment. The stock solution was prepared four times during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of the preliminary formulation trial and pilot DRF study (CiToxLAB study code: 13/195-100PE), an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.

- Concentration in vehicle: The test item was formulated in the aqueous ammonium-chloride buffer as a stock solution at the concentration of 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurred. All pairs mated within the first 7 days from the onset of pairing.
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (and as Day 0 of pregnancy).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle was assessed in the concentration range of 5 mg/mL-200 mg/mL by Fourier-transformation infrared spectroscopy (FT-IR). According to the results the test item solution in aqueous ammonium-chloride buffer was stable over 7 days at room temperature and for 14 days when refrigerated (2-8ºC). (CiToxLAB Study code: 13/195-929AN).

Analysis of test item formulations was performed at the Test Site using a validated ICP method (ICP-AES) to determine the palladium content. The concentration measurements were performed on all preparations of the stock solutions and at the same time, from samples of diluted dose solutions. The sampling occasions coincided with the preparation of the stock solution.

Samples were taken from the test item formulations (including control) for concentration measurements. One set of samples was collected for analysis and one set retained as a back-up, for possible confirmatory analysis. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of the test item. No confirmatory analysis was required.

The measured concentrations of palladium in the formulations varied between 95.0 % and 111.9 % of the nominal. These results were within the range of acceptable values (85% - 115%).
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating period). They were then euthanized and subjected to necropsy examination.

Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum.
Frequency of treatment:
Once daily
Details on study schedule:
n/a
Remarks:
Doses / Concentrations:
4 mg/kg bw/day
Basis:
other: nominal dose
Remarks:
Doses / Concentrations:
20 mg/kg bw/day
Basis:
other: nominal dose
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
other: nominal dose
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director, based on available data, including the results of a dose range finding study (CiToxLAB study code 13/195-100PE), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
Positive control:
No
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Adult (parental) animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. The changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were performed once prior to first treatment (to allow for within-subject comparisons), and once a week thereafter. The animals were examined outside the home cage in a standard arena and at similar times on each occasion. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, twice a week thereafter and prior to necropsy.

Adult females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before necropsy).

The body weights of the females were also recorded on GD4, 10 and 17 in order to give accurate treatment volumes, however, these data were not evaluated statistically.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
Detailed histological examination was performed on the testes and epididymides in the control and high dose groups. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All animals were sacrificed on test day 29, after a dosing period of 28 days.
- Maternal animals: Dams were sacrificed following at least 4 days post-partum dosing.

GROSS NECROPSY
- Gross necropsy consisted of external appearance. The cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Special attention was paid to the organs of the reproductive system. The number of implantation sites and the number of corpora lutea were recorded. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination was performed on testes, epididymides and ovaries in the control and high dose groups. In addition, gross lesions (the stomachs of 9 males and 5 females from the High Dose) were also examined microscopically. The stomachs of two control males and females were also examined for comparison.

Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments as well as the epithelial capsule and ovarian stroma.
Postmortem examinations (offspring):
Pups euthanized at PND 4 were carefully examined externally for gross abnormalities. Dead pups were subjected to necropsy with macroscopic examination in order to identify the probable cause of death.
Statistics:
Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. When Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Chi2 test was performed when feasible.
Reproductive indices:
The following indices were calculated for each group:

Male mating index [%] = (Number of males with confirmed mating/Total number of males cohabited) x 100 [Measure of male's ability to mate]
Female mating index [%] = (Number of sperm-positive females/Total number of females cohabited) x 100 [Measure of female's ability to mate]

Male fertility index [%] = (Number of males impregnating a female/Total number of males cohabited) x 100 [Measure of male's ability to produce sperm that can fertilise eggs]
Female fertility index [%] = (Number of pregnant females/Number of sperm-positive females) x 100 [Measure of female's ability to become pregnant]

Gestation index [%] = (Number of females with live born pups/Number of pregnant females) x 100 [Measure of pregnancy that provides at least one live pup]
Offspring viability indices:
Survival index [%] = (Number of live pups (at designated time)/Number of pups born) x 100
Pre-implantation mortality [%] = (Number of Corpora lutea - Number of Implantations/Number of Corpora lutea) x 100
Intrauterine mortality [%] = (Number of implantations - Number of liveborns/Number of implantations) x 100
Total mortality [%] = (Number of implantations - Number of viable pups (PND4)/Number of implantations) x 100
% Males = (Number of pups examined - Number of males/Number of pups examined) x 100
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no clinical signs related to treatment. All animals were clinically normal.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of males treated at 100 mg/kg/day were slightly lower than controls from Week 1 onwards. This was due in part to the higher mean body weight for the control group. The differences from control attained statistical significance (p<0.01 on Days 7, 14, 21). Compared to the controls, mean value was approximately 7% lower on Day 27, and the difference was statistically significant (p<0.05).

Compared to the control, significantly lower body weight gain was noted for High dose males during Weeks 1 (p<0.01) and 3 (not statistically significant) resulting in lower overall (Days 0-27) gain value by 25% (p<0.05). In 5 of 12 males at 100 mg/kg/day suppressed body weight gain or slight body weight loss was recorded at the beginning of the treatment period (Days 0-3). For one male no body weight gain or a slight body weight loss was noted during the last week of the treatment. This animal had the most severe changes in the stomach in the form of mild inflammation and ulceration in the glandular stomach.

Body weight of males at 20 mg/kg/day was lower than control mean throughout the entire treatment period by 6-7%, and the differences were statistically significant (p<0.01 on days 7, 14 and p<0.05 on days 21 and 27). The body weight gain values of these males were significantly lower, than control mean during Weeks 1 (p<0.01) and 2 (not statistically significant). The overall (Days 0-27) body weight gain value was lower than the control mean by 22% (p<0.05). The individual body weight gain values were within the control range, with the exception of one male which had consistently suppressed body weight gain or slight body weight loss throughout the entire treatment period.

The mean body weight of males at 4 mg/kg/day did not differ significantly from the control mean.

It should be noted, that the mean body weight of control males was slightly higher (by approximately 2%) on Day 0 and the body weight values were in the higher range. In addition, two control males had overall body weight gain value higher than group mean by 25-30%.

Females at 100, 20 and 4 mg/kg/day had mean body weights and body weight gains comparable to the controls throughout the entire treatment period.

The average food consumption of males in all test item treated groups was lower, than control during Week 2 by approximately 12-13% at 4 and 100 mg/kg/day, and by 18% at 20 mg/kg/day. The differences attained statistical significance: p<0.01 for Low and Mid dose groups and p<0.05 for High Dose group, but were not clearly treatment related due to the lack of dose-response.

Lower than control mean food consumption was also recorded for all test item treated groups of males from the end of mating period up to Day 21. The differences were statistically significant (p<0.05) and attributed to the higher control value.

There were two males in the control group with food consumption well above the control mean.

The average food consumption in females in all test item treated groups was comparable with the control mean throughout the entire treatment period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test item formulations were found to be in the range of 95.0 and 111.9 % of of nominal concentrations and were therefore considered acceptable.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in Control and High Dose males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item related effects on organ weights. Occasional statistically significant differences in absolute testis and prostate weight were attributed to the lower body weights of the animals and therefore of no toxicologically significance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At necropsy, test item-related findings were observed at 100 mg/kg/day (High dose) in the form of focal/multifocal dark red discoloration of the glandular mucosa of the stomach in 9/12 males and 5/12 females.

Unilateral, focal, yellow/green discoloration of the epididymis was noted in 1/12 males in the High dose.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test item-related microscopic findings were observed in the glandular stomach of animals at 100 mg/kg/day (High dose). In all males with macroscopic changes (dark red discoloration), congestion was noted in the glandular mucosa of the stomach (9/9). In addition, 7/9 of these males had minimal to mild, mixed cellular infiltration, and minimal to mild mixed cellular inflammation was observed in 2/9 males. In one animal organizing ulcer in the glandular mucosa was found additionally. In 5/5 all High dose females congestion of the glandular mucosa of the stomach with minimal, mixed cellular infiltration was detected. The observation was in agreement with necropsy finding.

No test item-related microscopic findings were noted in the reproductive system of the males and females from the High dose group. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females. The primordial, secondary and tertiary follicles and corpora lutea were also bilaterally present.

The spermatogenic cells, representing different phases of the development and differentiation of the spermatozoons as well as interstitial cell structure were similar in Control and High Dose males.

The unilateral, focal, yellow/green discoloration of the epididymis in 1/12 males in the High dose was microscopically identified as moderate spermatocoele and was regarded as a background observation.

The other microscopic changes were regarded as incidental or background.
Dose descriptor:
NOAEL
Remarks:
fertility/reproductive parameters
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Decreased body weight gain at 20 and 100 mg/kg bw/day.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The number of viable pups on PND 0 and 4 was comparable for all groups including the control.
The incidence of mortality during 4 postnatal days was negligible and was 4 of 149 (Low dose), 5 of 146 (Mid dose), and 2 of 167 (High dose). No mortality occurred in control group.

CLINICAL SIGNS (OFFSPRING)
No effects

BODY WEIGHT (OFFSPRING)
There was no effect of treatment on the offspring body weight or body weight gain.

SEXUAL MATURATION (OFFSPRING)
Not examined

ORGAN WEIGHTS (OFFSPRING)
Not examined

GROSS PATHOLOGY (OFFSPRING)
No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.

HISTOPATHOLOGY (OFFSPRING)
Not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Viability, clinical signs, mortality, body weights, gross pathology
Reproductive effects observed:
not specified
Conclusions:
In an OECD Test Guideline 421 reproduction and developmental toxicity screening study, to GLP, parental rats (12/sex/group) were administered tetraamminepalladium dichloride by gavage at 0 (in aqueous ammonium-chloride), 4, 20 or 100 mg/kg bw/day. No adverse effects on reproductive parameters, or on development of offspring, were observed at any dose, resulting in a reproductive toxicity NOAEL of 100 mg/kg bw/day
Executive summary:

The potential of tetraammineplladium dichloride to adversely affect the fertility and reproductive parameters of rats was investigated in a reproductive and developmental screening study conducted according to OECD Test Guideline 421 and to GLP The test material was administered by oral gavage. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post mating). Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). Three dose groups (4, 20, and 100 mg/kg bw/day) and a control group were used, each containing 12 animals of each sex.

 

Parental (F0) animals were observed for clinical signs of toxicity throughout the study, with body weights and food consumption monitored. At necropsy, animals were subjected to external and internal macroscopic examinations for any abnormalities or pathological changes. Special attention was paid to the reproductive organs. The numbers of implantation sites and corpora lutea were recorded. Histopathological examination was performed on the ovaries, testes and epididymides of all animals in the control and high-dose groups, with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure. A number of reproductive indices were calculated from the collected data (including mating, fertility and gestation indices).

Effects on the glandular stomach (including discolouration, inflammation, and congestion) were seen in the high-dose animals, and likely reflects a local effect of treatment. These effects in the glandular stomach may have contributed to the significantly reduced body weight gain seen in males at 20 and 100 mg/kg bw/day. The no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 4 mg/kg/day.

 

No test item-related microscopic changes were noted in the reproductive organs, and there was no impact on fertility or on the measured reproductive parameters at any dose level. The NOAEL for reproductive toxicity was 100 mg/kg bw/day, the highest dose tested.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Overall, good-quality database which meets REACH Standard Information Requirements.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No relevant data in humans were identified.

However, a reliable reproduction/developmental screening toxicity study in rats has been conducted with tetraamminepalladium dichloride. Tetraamminepalladium dichloride is considered to fall within the scope of the read-across category "tetraamminepalladium salts". See IUCLID section 13 for full read-across justification report.

 

The potential of tetraamminepalladium dichloride to adversely affect the fertility and reproductive parameters of rats was investigated in a reproductive and developmental screening study conducted according to OECD Test Guideline 421 and to GLP. The test material was administered (in aqueous ammonium chloride buffer) by oral gavage. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post mating). Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). Three dose groups (4, 20, and 100 mg/kg bw/day) and a control group were used, each containing 12 animals of each sex.

 

Parental (F0) animals were observed for clinical signs of toxicity throughout the study, with body weights and food consumption monitored. At necropsy, animals were subjected to external and internal macroscopic examinations for any abnormalities or pathological changes. Special attention was paid to the reproductive organs. The numbers of implantation sites and corpora lutea were recorded. Histopathological examination was performed on the ovaries, testes and epididymides of all animals in the control and high-dose groups, with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure. A number of reproductive indices were calculated from the collected data (including mating, fertility and gestation indices).

 

Effects on the glandular stomach (including discolouration, inflammation, and congestion) were seen in the high-dose animals, and likely reflect a local effect of treatment. These effects in the glandular stomach may have contributed to the significantly reduced body weight gain seen in males at 20 and 100 mg/kg bw/day. The no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be 4 mg/kg bw/day.

 

No test item-related microscopic changes were noted in the reproductive organs, and there was no impact on fertility or on the measured reproductive parameters at any dose level. The NOAEL for reproductive toxicity was 100 mg/kg bw/day, the highest dose tested (Török-Bathó, 2015).



Justification for selection of Effect on fertility via oral route:
Reliable GLP study, conducted according to OECD guidelines, and the only reproduction/developmental toxicity study available.

Effects on developmental toxicity

Description of key information

In an OECD Test Guideline 421 reproduction and developmental toxicity screening study, to GLP, parental rats (12/sex/group) were administered tetraamminepalladium dichloride by gavage at 0 (in aqueous ammonium chloride), 4, 20 or 100 mg/kg bw/day. No adverse effects on the mothers, on reproductive parameters, or on development of offspring, were observed at any dose, resulting in a developmental toxicity NOAEL of 100 mg/kg bw/day (Török-Bathó, 2015).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
24 June 2014 - 09 August 2014 (last necropsy)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, conducted to GLP, on closely-related surrogate
Justification for type of information:
Within the category of tetraamminepalladium(II) compounds, data on three tetraamminepalladium(II) salts, the diacetate, dichloride, and hydrogen carbonate salts will be used to fill data gaps. Tetraamminepalladium(II) diacetate, dinitrate, dihydroxide and dichloride are the target substances within the group. In all substances covered, the palladium is in the 2+ oxidation state, co-ordinated to four neutral ammonia molecules (giving an overall 2+ charge on the complex). Thus, the difference in anion (acetate, nitrate, hydroxide, chloride or hydrogen carbonate) represents the only structural difference between the compounds in this group. As detailed in the read-across justification report (cfr IUCLID section 13), all the human health toxicity data included in the category member dossiers should be considered equally applicable to each of the category member substances.
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
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: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, from SPF colony
- Age at study initiation: Young adult rats, at least 10 weeks old at starting and at least 12 weeks at mating.
- Weight at study initiation: Males: 338 g – 399 g, Females: 219 g - 255 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment. - Fasting period before study: no data
- Housing: Type II and III polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany).
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany offered ad libitum
- Water (e.g. ad libitum): tap water from municipal supply as for human consumption from 500 ml bottle ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-23.8 °C (target range 22±3°C)
- Humidity (%): 40 - 70 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
Start of experiment (start of treatment): 24 June 2014
End of treatment: 22 July 2014 (males); 08 August 2014 (females)
End of experiment: 09 August 2014 (last necropsy)
Route of administration:
oral: gavage
Vehicle:
other: an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was used as the dosing vehicle.

The buffer was prepared as follows:
2.68 g ammonium chloride (Batch/Lot No.: BCBM5575V, Supplier: Sigma-Aldrich, Expiry: March 2015) was dissolved in 1000 ml distilled water (Batch/Lot No.: 0271113, Supplier: TEVA Pharmaceutical Works PLtd., Expiry: November 2016).
The pH was adjusted to 9 by adding approximately 1650-1850 µL of 29.1% ammonium hydroxide (Batch/Lot No.: SZBD2260V, Supplier: Sigma-Aldrich, Expiry: August 2015).

The first preparation was used for 7 consecutive days and was kept at room temperature. The next preparations were used for periods of up to 14 days, following confirmation of the stability, and the stock solution was kept refrigerated (2-8ºC). Dose solutions for treatment were diluted from the stock solution with aqueous ammonium-chloride buffer to the concentrations of 4.0 and 0.8 mg/mL, daily just before the treatment. The stock solution was prepared four times during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of the preliminary formulation trial and pilot DRF study (CiToxLAB study code: 13/195-100PE), an aqueous ammonium-chloride buffer (NH4Cl/NH4OH) was selected as the vehicle. The vehicle allowed formulation of a stable solution of the test item, considered suitable for the study purpose.

- Concentration in vehicle: The test item was formulated in the aqueous ammonium-chloride buffer as a stock solution at the concentration of 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle was assessed in the concentration range of 5 mg/mL-200 mg/mL by Fourier-transformation infrared spectroscopy (FT-IR). According to the results the test item solution in aqueous ammonium-chloride buffer was stable over 7 days at room temperature and for 14 days when refrigerated (2-8ºC). (CiToxLAB Study code: 13/195-929AN).

Analysis of test item formulations was performed at the Test Site using a validated ICP method (ICP-AES) to determine the palladium content. The concentration measurements were performed on all preparations of the stock solutions and at the same time, from samples of diluted dose solutions. The sampling occasions coincided with the preparation of the stock solution.

Samples were taken from the test item formulations (including control) for concentration measurements. One set of samples was collected for analysis and one set retained as a back-up, for possible confirmatory analysis. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of the test item. No confirmatory analysis was required.

The measured concentrations of palladium in the formulations varied between 95.0 % and 111.9 % of the nominal. These results were within the range of acceptable values (85% - 115%).
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Females remained with the same male until copulation occurred. All pairs mated within the first 7 days from the onset of pairing.
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (and as Day 0 of pregnancy).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post- mating period). They were then euthanized and subjected to necropsy examination.

Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) is defined as Day 0 post-partum.
Frequency of treatment:
Once daily
Duration of test:
At least 28 days (males); Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation (22-23 days) and up to and including the day before necropsy (at least 4 days post-partum dosing).
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director, based on available data, including the results of a dose range finding study (CiToxLAB study code 13/195-100PE), with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Adult (parental) animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical observations were made once a day.

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. The changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were performed once prior to first treatment (to allow for within-subject comparisons), and once a week thereafter. The animals were examined outside the home cage in a standard arena and at similar times on each occasion. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, twice a week thereafter and prior to necropsy.

Adult females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before necropsy).

The body weights of the females were also recorded on GD4, 10 and 17 in order to give accurate treatment volumes, however, these data were not evaluated statistically.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Dead pups and pups killed on day 4 post-partum were carefully examined externally for gross abnormalities

- External examinations: Yes: [all]. Any pups showing abnormalities in structure or behaviour will be subjected to necropsy with macroscopic examination
- Soft tissue examinations: Yes: gross only
- Skeletal examinations: Yes: gross only
- Head examinations: Yes: gross only
Statistics:
Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. When Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Chi2 test was performed when feasible.
Indices:
Reproductive indices

The following indices were calculated for each group:

Male mating index [%] = (Number of males with confirmed mating/Total number of males cohabited) x 100 [Measure of male's ability to mate]
Female mating index [%] = (Number of sperm-positive females/Total number of females cohabited) x 100 [Measure of female's ability to mate]

Male fertility index [%] = (Number of males impregnating a female/Total number of males cohabited) x 100 [Measure of male's ability to produce sperm that can fertilise eggs]
Female fertility index [%] = (Number of pregnant females/Number of sperm-positive females) x 100 [Measure of female's ability to become pregnant]

Gestation index [%] = (Number of females with live born pups/Number of pregnant females) x 100 [Measure of pregnancy that provides at least one live pup]

Offspring viability indices

Survival index [%] = (Number of live pups (at designated time)/Number of pups born) x 100
Pre-implantation mortality [%] = (Number of Corpora lutea - Number of Implantations/Number of Corpora lutea) x 100
Intrauterine mortality [%] = (Number of implantations - Number of liveborns/Number of implantations) x 100
Total mortality [%] = (Number of implantations - Number of viable pups (PND4)/Number of implantations) x 100
% Males = (Number of pups examined - Number of males/Number of pups examined) x 100
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:no effects
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
The number of viable pups on PND 0 and 4 was comparable for all groups including the control.
The incidence of mortality during 4 postnatal days was negligible and was 4 of 149 (Low dose), 5 of 146 (Mid dose), and 2 of 167 (High dose). No mortality occurred in control group.

CLINICAL SIGNS (OFFSPRING)
No effects

BODY WEIGHT (OFFSPRING)
There was no effect of treatment on the offspring body weight or body weight gain.

SEXUAL MATURATION (OFFSPRING)
Not examined

ORGAN WEIGHTS (OFFSPRING)
Not examined

GROSS PATHOLOGY (OFFSPRING)
No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.

HISTOPATHOLOGY (OFFSPRING)
Not examined
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryotoxicity, teratogenicity
Abnormalities:
not specified
Developmental effects observed:
no
Conclusions:
In an OECD Test Guideline 421 reproduction and developmental toxicity screening study, to GLP, parental rats (12/sex/group) were administered tetraamminepalladium dichloride by gavage at 0 (in aqueous ammonium-chloride), 4, 20 or 100 mg/kg bw/day. No adverse effects on maternal toxicity, reproductive parameters, or on development of offspring, were observed at any dose, resulting in a NOAEL of 100 mg/kg bw/day
Executive summary:

The potential of a solution of Tetraamminepalladium dichloride to adversely affect the development of rat pups was investigated in a reproductive and developmental screening study conducted according to OECD Test Guideline 421 and to GLP. The test material was administered (in aqueous ammonium-chloride buffer) by oral gavage. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post mating). Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). Three dose groups (4, 20, and 100 mg/kg bw/day) and a control group were used, each containing 12 animals of each sex.

 

Parental (F0) animals were observed for clinical signs of toxicity throughout the study, with body weights and food consumption monitored. At necropsy, animals were subjected to external and internal macroscopic examinations for any abnormalities or pathological changes. Special attention was paid to the reproductive organs. The numbers of implantation sites and corpora lutea were recorded. Histopathologic examination was performed on the ovaries, testes and epididymides of all animals in the control and high-dose groups, with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure. On postnatal day 4, pups were carefully examined for gross abnormalities at necropsy.

 

There were no signs of maternal toxicity or developmental toxicity effect at any dose level. In conclusion, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for developmental toxicity was considered to be 100 mg/kg bw/day (the highest tested dose).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Overall, good-quality database which meets REACH Standard Information Requirements.
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

No relevant data in humans were identified.

 

However, a reliable reproduction/developmental screening toxicity study in rats has been conducted with tetraamminepalladium dichloride. Tetraamminepalladium dichloride is considered to fall within the scope of the read-across category "tetraamminepalladium salts". See IULCID section 13 for full read-across justification report.

 

The potential of a solution of tetraamminepalladium dichloride to adversely affect the development of rat pups was investigated in a reproductive and developmental screening study conducted according to OECD Test Guideline 421, and to GLP. The test material was administered (in aqueous ammonium chloride buffer) by oral gavage. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post mating). Females were dosed for 14 days pre-mating, for up to 7 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). Three dose groups (4, 20, and 100 mg/kg bw/day) and a control group were used, each containing 12 animals of each sex.

 

Parental (F0) animals were observed for clinical signs of toxicity throughout the study, with body weights and food consumption monitored. At necropsy, animals were subjected to external and internal macroscopic examinations for any abnormalities or pathological changes. Special attention was paid to the reproductive organs. The numbers of implantation sites and corpora lutea were recorded. Histopathologic examination was performed on the ovaries, testes and epididymides of all animals in the control and high-dose groups, with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure. On postnatal day 4, pups were carefully examined for gross abnormalities at necropsy.

 

There were no signs of maternal toxicity or developmental toxicity effects at any dose level. In conclusion, under the conditions of this study, the NOAEL for developmental toxicity was considered to be 100 mg/kg bw/day (the highest tested dose) (Török-Bathó, 2015).



Justification for selection of Effect on developmental toxicity: via oral route:
Reliable GLP study, conducted according to OECD guidelines, and the only reproduction/developmental toxicity study available.

Toxicity to reproduction: other studies

Description of key information

No data identified

Additional information

No data identified.

Justification for classification or non-classification

No adverse effects on reproductive parameters (sexual function or fertility) or development of offspring were seen in a reliable guideline screening assay with tetraamminepalladium dichloride. As such, classification for reproductive toxicity is not required, according to EU CLP criteria (EC 1272/2008).

Additional information