Palladium dichloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No data identified

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June 2014 to 23 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 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, approximately 11 weeks old at starting and 13 weeks at mating
- Weight at study initiation: Males: 355 g – 413 g, Females: 222 g - 255 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
- Fasting period before study:
- Housing: Type II and/or III polypropylene/polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Rodents were group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- 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 ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 24.6 °C (target range 22±3°C)
- Humidity (%): 33 – 66 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily

IN-LIFE DATES: From: 10 June 2014 To: 26 July 2014 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance has low solubility in water (0.000001 g/l)
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml (for control, low-, mid-, and high dose levels)
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): MKBQ9948V / MKBP7039V

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days (all pairs mated within the first 5 days of the mating period) .
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- 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:

The test item was formulated in the vehicle, as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared weekly or biweekly, based on the stability assessment results. Stability of the test item in the selected vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to 13/193-929AN).

Analysis of Palladium dihydroxide formulation samples of 1 – 200 mg/mL concentration range showed no decrease of concentration and was consideredacceptable stabilityle for 14 days at room temperature. The infrared spectra both in the liquid phase and the solid phase (in CsI disk) showed no presence of degradation products and support the stability of the test item in corn oil for 14 days (according to 13/193-929AN).

Analysis of test item formulations for concentration and homogeneity was performed in the Seibersdorf Labor GmbH. Top, middle and bottom duplicate samples were taken and analysed from test item formulations and all concentrations. Sample analysis was made on 2 occasions for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test item.

Formulations prepared on Day 0 (for use over 7 days), were sampled and sent to the Test Site for rapid formulation analysis to provide confirmation on formulation concentration and homogeneity. All other formulations were sent for analysis at the end of the study as agreed with the Sponsor.

Description of the analytical method and the results of the formulation analysis are included in an analytical Phase report, provided by the Principal Investigator in Appendix 4 of the study report.

Duration of treatment / exposure:

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day 4

Frequency of treatment:

Test item or negative control treated animals were administered the dosing formulations daily on a 7 days/week basis

Details on study schedule:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 13/193-100PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

12 animals/sex/group, 4 groups (48 male, 48 female rats)

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 and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 13/193-100PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.

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

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. 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 were 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, at least weekly thereafter and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weight of the female animals was additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly (on the days of body weight measurement).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

The following parameters parameters were also evaluated, as detailed in 7.5.1 Repeated dose toxicity: oral:
Haematology
Clinical chemistry
Urinalysis
Neurobehavioural examination

Oestrous cyclicity (parental animals):

Oestrous cycle assessed during the mating period, until a sperm positive vaginal smear or a vaginal plug was identified

Sperm parameters (parental animals):

Testis sperm content assessed. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure (see 'Postmortem exaimnations (Paranteral animals)' below for further details).

Litter observations:

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Data were recorded to provide information on:
- Mean pup body weight (per pup within the group and per litter) on PND 0 and 4
- Mean pup body weight gain (per litter) between postnatal Days 0-4
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0 and 4
- Survival Index of pups on postnatal Days 0 and 4
- Sex ratio % (on postnatal Days 0 and 4)

Postmortem examinations (parental animals):

Surviving adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.

Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Special attention was paid to the organs of the reproductive system. Notably, a detailed histological examination of the reproductive organs of all animals of the control and high dose group and of all males (testes, epididymides, prostate gland, seminal vesicles with coagulation gland and preputial gland) that failed to sire and all females (uterus, cervix, ovary, oviduct and vagina for females) that failed to deliver healthy pups.

The number of implantation sites and of corpora lutea were recorded in the females as applicable.

The weight of a range of organs was determined, including those of the reproductive system (including the ovaries and uterus (including cervix) for the females, and the testis, epididymis, seminal vesicles and prostrate gland weights for the males).

Postmortem examinations (offspring):

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). 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. For a 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 as feasible.

Reproductive indices:

See Table below: Formulas for Calculation of Mating and Fertility Indices

Offspring viability indices:

See Table below: Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

No test-item related adverse effects on reproductive performance (notably mating and fertility indices, and gestation index), oestrous cyclicity or sperm parameters, were observed. The numbers of implantation sites and corpora lutea were also unaffected by treatment. No treatment-related effect on the weight, gross appearance or microscopic examination of the reproductive organs was reported.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or on necropsy observations.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

gross pathology

other: developmental effects

Reproductive effects observed:

not specified

Conclusions:

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of palladium dihydroxide for at least 28 days, the systemic and reproductive NOAEL was the highest tested dose (1000 mg/kg bw/day)

Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, rats were orally administered palladium dihydroxide by stomach tube (gavage) at about 0, 100, 300 or 1000 mg/kg bw/day. Male and female Wistar rats (12 animals/sex/group) were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were also treated throughout gestation and up to and including postpartum/lactation Day 4.

No test-item related adverse effects on reproductive performance (notably mating and fertility indices, and gestation index), oestrous cyclicity or sperm parameters, were observed. The numbers of implantation sites and corpora lutea were also unaffected by treatment. Special attention was paid to the organs of the reproductive system. No treatment-related effect on the weight, gross appearance or microscopic examination of the reproductive organs was reported.

In conclusion, under the conditions of this study, the systemic and reproductive toxicity no-observed-adverse-effect level (NOAEL) of palladium dihydroxide was 1000 mg/kg bw/day (the highest tested dose).

Reference 2

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:

10 June 2014 to 23 July 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP

Justification for type of information:

Palladium dihydroxide is considered to fall within the scope of the read-across category "uncomplexed and partially-complexed palladium compounds". See section 13 for full justification report.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 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, approximately 11 weeks old at starting and 13 weeks at mating
- Weight at study initiation: Males: 355 g – 413 g, Females: 222 g - 255 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
- Fasting period before study:
- Housing: Type II and/or III polypropylene/polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Rodents were group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- 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 ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 24.6 °C (target range 22±3°C)
- Humidity (%): 33 – 66 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily

IN-LIFE DATES: From: 10 June 2014 To: 26 July 2014 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance has low solubility in water (0.000001 g/l)
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml (for control, low-, mid-, and high dose levels)
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): MKBQ9948V / MKBP7039V

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days (all pairs mated within the first 5 days of the mating period) .
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- 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:

The test item was formulated in the vehicle, as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared weekly or biweekly, based on the stability assessment results. Stability of the test item in the selected vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to 13/193-929AN).

Analysis of Palladium dihydroxide formulation samples of 1 – 200 mg/mL concentration range showed no decrease of concentration and was consideredacceptable stabilityle for 14 days at room temperature. The infrared spectra both in the liquid phase and the solid phase (in CsI disk) showed no presence of degradation products and support the stability of the test item in corn oil for 14 days (according to 13/193-929AN).

Analysis of test item formulations for concentration and homogeneity was performed in the Seibersdorf Labor GmbH. Top, middle and bottom duplicate samples were taken and analysed from test item formulations and all concentrations. Sample analysis was made on 2 occasions for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test item.

Formulations prepared on Day 0 (for use over 7 days), were sampled and sent to the Test Site for rapid formulation analysis to provide confirmation on formulation concentration and homogeneity. All other formulations were sent for analysis at the end of the study as agreed with the Sponsor.

Description of the analytical method and the results of the formulation analysis are included in an analytical Phase report, provided by the Principal Investigator in Appendix 4 of the study report.

Duration of treatment / exposure:

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day 4

Frequency of treatment:

Test item or negative control treated animals were administered the dosing formulations daily on a 7 days/week basis

Details on study schedule:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available data and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 13/193-100PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

12 animals/sex/group, 4 groups (48 male, 48 female rats)

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 and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 13/193-100PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.

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

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. 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 were 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, at least weekly thereafter and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weight of the female animals was additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly (on the days of body weight measurement).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

The following parameters parameters were also evaluated, as detailed in 7.5.1 Repeated dose toxicity: oral:
Haematology
Clinical chemistry
Urinalysis
Neurobehavioural examination

Oestrous cyclicity (parental animals):

Oestrous cycle assessed during the mating period, until a sperm positive vaginal smear or a vaginal plug was identified

Sperm parameters (parental animals):

Testis sperm content assessed. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure (see 'Postmortem exaimnations (Paranteral animals)' below for further details).

Litter observations:

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Data were recorded to provide information on:
- Mean pup body weight (per pup within the group and per litter) on PND 0 and 4
- Mean pup body weight gain (per litter) between postnatal Days 0-4
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0 and 4
- Survival Index of pups on postnatal Days 0 and 4
- Sex ratio % (on postnatal Days 0 and 4)

Postmortem examinations (parental animals):

Surviving adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.

Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Special attention was paid to the organs of the reproductive system. Notably, a detailed histological examination of the reproductive organs of all animals of the control and high dose group and of all males (testes, epididymides, prostate gland, seminal vesicles with coagulation gland and preputial gland) that failed to sire and all females (uterus, cervix, ovary, oviduct and vagina for females) that failed to deliver healthy pups.

The number of implantation sites and of corpora lutea were recorded in the females as applicable.

The weight of a range of organs was determined, including those of the reproductive system (including the ovaries and uterus (including cervix) for the females, and the testis, epididymis, seminal vesicles and prostrate gland weights for the males).

Postmortem examinations (offspring):

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). 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. For a 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 as feasible.

Reproductive indices:

See Table below: Formulas for Calculation of Mating and Fertility Indices

Offspring viability indices:

See Table below: Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

No test-item related adverse effects on reproductive performance (notably mating and fertility indices, and gestation index), oestrous cyclicity or sperm parameters, were observed. The numbers of implantation sites and corpora lutea were also unaffected by treatment. No treatment-related effect on the weight, gross appearance or microscopic examination of the reproductive organs was reported.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or on necropsy observations.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

gross pathology

other: developmental effects

Reproductive effects observed:

not specified

Conclusions:

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of palladium dihydroxide for at least 28 days, the systemic and reproductive NOAEL was the highest tested dose (1000 mg/kg bw/day)

Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, rats were orally administered palladium dihydroxide by stomach tube (gavage) at about 0, 100, 300 or 1000 mg/kg bw/day. Male and female Wistar rats (12 animals/sex/group) were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were also treated throughout gestation and up to and including postpartum/lactation Day 4.

No test-item related adverse effects on reproductive performance (notably mating and fertility indices, and gestation index), oestrous cyclicity or sperm parameters, were observed. The numbers of implantation sites and corpora lutea were also unaffected by treatment. Special attention was paid to the organs of the reproductive system. No treatment-related effect on the weight, gross appearance or microscopic examination of the reproductive organs was reported.

In conclusion, under the conditions of this study, the systemic and reproductive toxicity no-observed-adverse-effect level (NOAEL) of palladium dihydroxide was 1000 mg/kg bw/day (the highest tested dose).

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:

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 reproduction data on palladium dichloride were identified.

However, a reliable combined repeated dose toxicity and reproduction/developmental screening study in rats has been conducted with palladium dihydroxide. Palladium dihydroxide is considered to fall within the scope of the read-across category "uncomplexed and partially-complexed palladium compounds". See section 13 for full justification report.

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, rats were orally administered palladium dihydroxide by stomach tube (gavage) at about 0, 100, 300 or 1000 mg/kg bw/day. Male and female Wistar rats (12 animals/sex/group) were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were also treated throughout gestation and up to and including postpartum/lactation Day 4 (i.e. around 50 days in total). No test-item related adverse effects on reproductive performance (notably mating and fertility indices, and gestation index), oestrous cyclicity or sperm parameters, were observed.The numbers of implantation sites and corpora lutea were also unaffected by treatment. No treatment-related effect on the weight, gross appearance or microscopic examination of the reproductive organs was reported. In conclusion, under the conditions of this study, the systemic and reproductive toxicity NOAEL of palladium dihydroxide was 1000 mg/kg bw/day (the highest tested dose) (Török-Bathó, 2015).  

 

No reproductive toxicity studies by the inhalation or dermal route were identified, or are required.

Short description of key information:
In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of palladium dihydroxide, the systemic and reproductive NOAEL was the highest tested dose (1000 mg/kg bw/day) (Török-Bathó, 2015).

Justification for selection of Effect on fertility: via oral route:
GLP study, conducted according to OECD guidelines.

Effects on developmental toxicity

Description of key information

No data identified.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June 2014 to 23 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 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, approximately 11 weeks old at starting and 13 weeks at mating
- Weight at study initiation: Males: 355 g – 413 g, Females: 222 g - 255 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
- Fasting period before study:
- Housing: Type II and/or III polypropylene/polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Rodents were group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- 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 ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 24.6 °C (target range 22±3°C)
- Humidity (%): 33 – 66 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily

IN-LIFE DATES: From: 10 June 2014 To: 26 July 2014 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance has low solubility in water (0.000001 g/l)
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml (for control, low-, mid-, and high dose levels)
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): MKBQ9948V / MKBP7039V
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was formulated in the vehicle, as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared weekly or biweekly, based on the stability assessment results. Stability of the test item in the selected vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to 13/193-929AN).

Analysis of Palladium dihydroxide formulation samples of 1 – 200 mg/mL concentration range showed no decrease of concentration and was consideredacceptable stabilityle for 14 days at room temperature. The infrared spectra both in the liquid phase and the solid phase (in CsI disk) showed no presence of degradation products and support the stability of the test item in corn oil for 14 days (according to 13/193-929AN).

Analysis of test item formulations for concentration and homogeneity was performed in the Seibersdorf Labor GmbH. Top, middle and bottom duplicate samples were taken and analysed from test item formulations and all concentrations. Sample analysis was made on 2 occasions for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test item.

Formulations prepared on Day 0 (for use over 7 days), were sampled and sent to the Test Site for rapid formulation analysis to provide confirmation on formulation concentration and homogeneity. All other formulations were sent for analysis at the end of the study as agreed with the Sponsor.

Description of the analytical method and the results of the formulation analysis are included in an analytical Phase report, provided by the Principal Investigator in Appendix 4 of the study report.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days (all pairs mated within the first 5 days of the mating period).
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day 4

Frequency of treatment:

Test item or negative control treated animals were administered the dosing formulations daily on a 7 days/week basis

Duration of test:

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day 4.

No. of animals per sex per dose:

12 animals/sex/group, 4 groups (48 male, 48 female rats)

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 and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 13/193-100PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.

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

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. 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 were 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, at least weekly thereafter and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weight of the female animals was additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly (on the days of body weight measurement).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

The following parameters parameters were also evaluated, as detailed in 7.5.1 Repeated dose toxicity: oral:
Haematology
Clinical chemistry
Urinalysis
Neurobehavioural examination

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post-natal day 5
- Organs examined:
Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the females as applicable.

For the adult animals, detailed histological examination was performed as follows:
• on the selected list (Table 1, below) of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• all macroscopic findings (abnormalities), except of minor order from all animals
• reproductive organs of all animals of the control and high dose group and of all females (uterus, cervix, ovary, oviduct and vagina) that failed to deliver healthy pups.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No. Uterus (including cervix) was weighed at terminal sacrifice at PND 5
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

The F1 offspring were assessed for number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups), and to detect the presence of gross abnormalities. In addition, pups were monitored for any behavioural changes. Live pups were counted, sexed and weighed individually within 24 hours of parturition (post-natal day (PND)0 or PND1) and on PND4. All the litters were checked daily for the number of viable and dead pups. Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). 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. For a 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 as feasible.

Indices:

A number of offspring viability and sex ratio indices were calculated (including survival index, pre-implantation and intrauterine mortality, and sex ratio of males). As detailed in Table 2, below

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or on necropsy observations.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental effects

Developmental effects observed:

no

Conclusions:

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of palladium dihydroxide for at least 28 days, the systemic and developmental toxicity NOAEL was the highest tested dose (1000 mg/kg bw/day)

Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, rats were orally administered palladium dihydroxide by stomach tube (gavage) at about 0, 100, 300 or 1000 mg/kg bw/day. Male and female Wistar rats (12 animals/sex/group) were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were also treated throughout gestation and up to and including postpartum/lactation Day 4.

 

No maternal systemic toxicity was observed, and the numbers of implantation sites and corpora lutea were unaffected by treatment.

 

The F1 offspring were assessed for number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups), and to detect the presence of gross abnormalities. A number of offspring viability and sex ratio indices were calculated (including survival index, pre-implantation and intrauterine mortality, and sex ratio of males). In addition, pups were monitored for any behavioural changes. Live pups were counted, sexed and weighed individually within 24 hours of parturition (post-natal day (PND)0 or PND1) and on PND4. All the litters were checked daily for the number of viable and dead pups. Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death. No foetal toxicity or developmental effects were reported.

In conclusion, under the conditions of this study, no treatment-related effects on maternal/foetal toxicity, or developmental effects, were seen resulting in a systemic and developmental toxicity no-observed-adverse-effect level (NOAEL) for palladium dihydroxide of 1000 mg/kg bw/day (the highest tested dose).

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

10 June 2014 to 23 July 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, to GLP

Justification for type of information:

Palladium dihydroxide is considered to fall within the scope of the read-across category "uncomplexed and partially-complexed palladium compounds". See section 13 for full justification report.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the 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, approximately 11 weeks old at starting and 13 weeks at mating
- Weight at study initiation: Males: 355 g – 413 g, Females: 222 g - 255 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
- Fasting period before study:
- Housing: Type II and/or III polypropylene/polycarbonate cages. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Rodents were group-housed, up to 4 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
- 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 ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 – 24.6 °C (target range 22±3°C)
- Humidity (%): 33 – 66 % (target range 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily

IN-LIFE DATES: From: 10 June 2014 To: 26 July 2014 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The substance has low solubility in water (0.000001 g/l)
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml (for control, low-, mid-, and high dose levels)
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): MKBQ9948V / MKBP7039V
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was formulated in the vehicle, as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared weekly or biweekly, based on the stability assessment results. Stability of the test item in the selected vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use, according to 13/193-929AN).

Analysis of Palladium dihydroxide formulation samples of 1 – 200 mg/mL concentration range showed no decrease of concentration and was consideredacceptable stabilityle for 14 days at room temperature. The infrared spectra both in the liquid phase and the solid phase (in CsI disk) showed no presence of degradation products and support the stability of the test item in corn oil for 14 days (according to 13/193-929AN).

Analysis of test item formulations for concentration and homogeneity was performed in the Seibersdorf Labor GmbH. Top, middle and bottom duplicate samples were taken and analysed from test item formulations and all concentrations. Sample analysis was made on 2 occasions for homogeneity (top, mid and bottom) and all prepared formulations were analysed for concentration. One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the Group 1 (control) solution to confirm the absence of test item.

Formulations prepared on Day 0 (for use over 7 days), were sampled and sent to the Test Site for rapid formulation analysis to provide confirmation on formulation concentration and homogeneity. All other formulations were sent for analysis at the end of the study as agreed with the Sponsor.

Description of the analytical method and the results of the formulation analysis are included in an analytical Phase report, provided by the Principal Investigator in Appendix 4 of the study report.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 5 days (all pairs mated within the first 5 days of the mating period).
- Proof of pregnancy: The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day 4

Frequency of treatment:

Test item or negative control treated animals were administered the dosing formulations daily on a 7 days/week basis

Duration of test:

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation Day 4.

No. of animals per sex per dose:

12 animals/sex/group, 4 groups (48 male, 48 female rats)

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 and information from previous experimental work, including the results of a repeated dose range finding study in the rat (CiToxLAB study code 13/193-100PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The selected highest dose of 1000 mg/kg bw/day is a limit dose for this study type.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.

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

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. 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 were 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, at least weekly thereafter and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on post partum Days PPD0 (within 24 hours after parturition), PPD4 and before termination. Body weight of the female animals was additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION:
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly (on the days of body weight measurement).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

The following parameters parameters were also evaluated, as detailed in 7.5.1 Repeated dose toxicity: oral:
Haematology
Clinical chemistry
Urinalysis
Neurobehavioural examination

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post-natal day 5
- Organs examined:
Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the females as applicable.

For the adult animals, detailed histological examination was performed as follows:
• on the selected list (Table 1, below) of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
• all macroscopic findings (abnormalities), except of minor order from all animals
• reproductive organs of all animals of the control and high dose group and of all females (uterus, cervix, ovary, oviduct and vagina) that failed to deliver healthy pups.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No. Uterus (including cervix) was weighed at terminal sacrifice at PND 5
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

The F1 offspring were assessed for number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups), and to detect the presence of gross abnormalities. In addition, pups were monitored for any behavioural changes. Live pups were counted, sexed and weighed individually within 24 hours of parturition (post-natal day (PND)0 or PND1) and on PND4. All the litters were checked daily for the number of viable and dead pups. Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest). 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. For a 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 as feasible.

Indices:

A number of offspring viability and sex ratio indices were calculated (including survival index, pre-implantation and intrauterine mortality, and sex ratio of males). As detailed in Table 2, below

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no adverse effects ascribed to test item administration on the F1 offspring viability, clinical signs, development or on necropsy observations.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental effects

Developmental effects observed:

no

Conclusions:

In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of palladium dihydroxide for at least 28 days, the systemic and developmental toxicity NOAEL was the highest tested dose (1000 mg/kg bw/day)

Executive summary:

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, rats were orally administered palladium dihydroxide by stomach tube (gavage) at about 0, 100, 300 or 1000 mg/kg bw/day. Male and female Wistar rats (12 animals/sex/group) were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were also treated throughout gestation and up to and including postpartum/lactation Day 4.

 

No maternal systemic toxicity was observed, and the numbers of implantation sites and corpora lutea were unaffected by treatment.

 

The F1 offspring were assessed for number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups), and to detect the presence of gross abnormalities. A number of offspring viability and sex ratio indices were calculated (including survival index, pre-implantation and intrauterine mortality, and sex ratio of males). In addition, pups were monitored for any behavioural changes. Live pups were counted, sexed and weighed individually within 24 hours of parturition (post-natal day (PND)0 or PND1) and on PND4. All the litters were checked daily for the number of viable and dead pups. Dead pups and pups euthanized at PND 4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death. No foetal toxicity or developmental effects were reported.

In conclusion, under the conditions of this study, no treatment-related effects on maternal/foetal toxicity, or developmental effects, were seen resulting in a systemic and developmental toxicity no-observed-adverse-effect level (NOAEL) for palladium dihydroxide of 1000 mg/kg bw/day (the highest tested dose).

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:

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 developmental toxicity data on palladium dichloride were identified.  

However, a reliable combined repeated dose toxicity and reproduction/developmental screening study in rats has been conducted with palladium dihydroxide.Palladium dihydroxide is considered to fall within the scope of the read-across category "uncomplexed and partially-complexed palladium compounds". See section 13 for full justification report

In a combined repeated dose toxicity and reproductive/developmental toxicity screening study, conducted according to OECD Test Guideline 422 and to GLP, rats were orally administered palladium dihydroxide by stomach tube (gavage) at about 0, 100, 300 or 1000 mg/kg bw/day. Male and female Wistar rats (12 animals/sex/group) were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were also treated throughout gestation and up to and including postpartum/lactation Day 4 (i.e. around 50 days in total). No maternal systemic toxicity was observed, and the numbers of implantation sites and corpora lutea were unaffected by treatment.The F1 offspring were assessed for number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups), and to detect the presence of gross abnormalities. A number of offspring viability and sex ratio indices were calculated (including survival index, pre-implantation and intrauterine mortality, and sex ratio of males). In addition, pups were monitored for any behavioural changes. Live pups were counted, sexed and weighed individually within 24 hours of parturition (post-natal day (PND)0 or PND1) and on PND4. All the litters were checked daily for the numbers of viable and dead pups. Dead pups and pups euthanized at PND4 were examined externally for gross abnormalities. Dead pups were necropsied with macroscopic examination in order to identify the probable cause of death. No foetal toxicity or developmental effects were reported.In conclusion, under the conditions of this study, no treatment-related effects on maternal/foetal toxicity, or developmental effects, were seen resulting in a systemic and developmental toxicity NOAEL for palladium dihydroxide of 1000 mg/kg bw/day (the highest tested dose) (Török-Bathó, 2015).

No developmental toxicity studies by the inhalation or dermal route were identified, or are required.

Short description of key information:
In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, involving the gavage administration of palladium dihydroxide, the systemic and developmental NOAEL was the highest tested dose (1000 mg/kg bw/day) (Török-Bathó, 2015).

Justification for selection of Effect on developmental toxicity: via oral route:
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 combined repeated dose toxicity study and reproductive/developmental toxicity screening study with palladium dihydroxide. As such, classification for reproductive toxicity for palladium dichloride is not required, according to EU CLP criteria (EC 1272/2008).