Diammonium hexachloropalladate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2014 to 3 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study to GLP (with no deviations)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

19168-23-1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Diammonium hexachloropalladate
- Substance type: no data
- Physical state: red powder
- Analytical purity: 30.09% (Pd)
- Impurities (identity and concentrations): Au (11 ppm); Ca (4 ppm); Fe (1 ppm); K (45 ppm); Na (40 ppm); Rh (1 ppm).
- Lot/batch No.: DZ0192
- Expiration date of the lot/batch: 07 July 2018
- Stability under test conditions: 4 hours (room temperature)
- Storage condition of test material: Room temperature (15-25 deg C, below 70% relative humidity)

Test animals

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
- Age at study initiation: Young adult rats, approximately 10 weeks old at start and at least 12 weeks at mating.
- Weight at study initiation: Males: 362 g – 455 g, Females: 235 g - 279 g
- 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): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6-24.5 °C (target range 22±3°C)
- Humidity (%): 33 - 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: From: 18 July 2014 To: 03 September 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly.

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the formulation and preliminary analytical trials and in agreement with the Sponsor.
- Concentration in vehicle: 2, 6 and 20 mg/ml for low, mid and high doses
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): MKBP7039V/MKBQ9948V
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: One female was housed with one male from the same dose group (1:1 mating) in a single cage.
- Length of cohabitation: Females remained with the same male until copulation occured.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

All pairs mated within the first 4 days from the onset of pairing, except female nr. 2512, whose original male (nr. 2012) was found dead at the beginning of the pre-mating period (on Day 4). This animal was replaced in order to ensure sufficient number of litters.

Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item content in the dose formulations was determined based on palladium content measured by ICP-AES analysis seven times during the study.
No test item was detected in the control solution samples. The test item formulations were found to be in the range of 66.5 and 95.5 % of the nominal concentrations in the low dose group (10 mg/kg bw/day), 82.8 and 100.9% in the mid dose group (30 mg/kg bw/day), and 92.1 and 101.2% in the high dose group. The mean exposure during the experiment is calculated as 81%, 91% and 98% of the targeted in the low, mid and high dose groups respectively. Therefore, the achieved dose intake was 90% for the mid and high dose formulations and 85% for the low dose formulation. The formulation acceptance criteria is generally +/- 15% of the nominal for suspensions. As the NOAEL is higher than the lowest dose, this deviation has no impact on the outcome of the study.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating). They were then euthanized and subjected to necropsy examination.

Females were dosed for 14 days pre-mating, for up to 4 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 (i.e. when parturition was complete) was defined as Day 0 post-partum.

Frequency of treatment:

The test item was administered daily by oral gavage on a 7 days/week basis.

Details on study schedule:

n/a

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0
Basis:
other: nominal: gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
10 mg/kg bw/day
Basis:
other: nominal: gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
other: nominal: gavage

Doses / concentrations 4

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
other: nominal: gavage

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 13/194-100PE, ref. 8) and a 28 days toxicity study (CiToxLAB study code 13/194-100P, ref 10). In the dose range finding study, administration of 600 mg/kg bw/day caused severe signs of toxicity (body weight loss, very low food consumption and marked changes of the gastric mucosa) and led to the death of half of the treated animals and euthanasia of the remaining animals by day 5. Similar signs were observed at 300 mg/kg bw/day but at a lower severity; this dose was also considered to be higher than the maximum tolerated and all animals were terminated prematurely (after a maximum of 6 doses). Administration of 100 mg/kg bw/day caused changes in the gastric mucosa but no effect on clinical condition, body weight, food consumption or organ weights. Based on these results and particularly the changes in the gastric mucosa due to contact with the test item, the highest dose level for the OECD 421 study was selected to be 100 mg/kg bw/day; a higher dose was considered likely to cause toxic effects and possible suffering. The lowest dose selected was expected to be a NOAEL.
- Rationale for animal assignment (if not random): radomisation based on body weight

Positive control:

No positive control

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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. Any changes were recorded including onset, degree and duration of signs as applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were made once prior to first treatment (to allow for within-subject comparisons), and once a week thereafter.

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, stereotypes (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
- Time schedule for examinations:
All adult animals were weighed with an accuracy of 1 g for randomization purposes, then on Day 0, weekly thereafter and at termination.

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 termination).

Body weight of the females was additionally weighed on GD4, 10 and 17 in order to give accurate treatment volumes but these data were not evaluated statistically.


OTHER:
Food consumption measurement: Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 and then weekly thereafter.

Observation of the delivery process, offspring and nursing instinct: Females were allowed to litter and rear their offspring. The delivery process was observed as "carefully as possible".

The duration of gestation was recorded and was calculated from day 0 of pregnancy and until completion of parturition.
Dams were observed for signs of nest building and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach.

Oestrous cyclicity (parental animals):

Not assessed

Sperm parameters (parental animals):

[See 'Postmortem examinations (Parental animals)' section below for details]

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are smaller than normal pups) and to detect the presence of gross abnormalities.

Live pups were counted, sexed, weighed individually within 24 hours of parturition (on the first day after parturition was complete, i.e. post partal PPD/ post natal day PND0 or 1) and on PND4 with an accuracy of 0.01g.

All the litters were checked daily for the number of viable and dead pups. The pups which were found dead were subjected to necropsy with macroscopic examination. Some of the pups found dead were cannibalised. They were counted and the sex determined but were not further examined.

- 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):

Gross necropsy was performed on each animal irrespective of the date of death. Terminally (one day after the last treatment), animals were sacrificed under pentobarbital anaesthesia by exsanguination. The external appearance was examined. The cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were 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 the number of corpora lutea were recorded.

At the time of termination, body weight and the weight of the following organs from all parental animals were determined:

- With a precision of at least 0.01 g: uterus (with cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain
- With a precision of at least 0.001 g: ovaries

Testes and epididymides were weighed individually. Individual and/or paired absolute organ weights were reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised.
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution.

The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Detailed histological examination was performed on testes, epididymides and ovaries in the control and high dose groups. In addition gross lesions (the stomachs of 12 males and 1 female from the High Dose, the spleens from 1 control, 1 low dose and 1 high dose female) were also examined microscopically.

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 of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

Pups euthanized on PND 4 were examined externally for gross abnormalities. Dead pups were subjected to necropsy and 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. 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 when feasible.

Reproductive indices:

Male Mating Index [%] = (No. of males with confirmed mating/Total no. of males cohabited) x 100 :-[Measure of male's ability to mate]
Female Mating Index [%] = (No. of sperm-positive females/Total no. of females cohabited) x 100 :-[Measure of female's ability to mate]

Male Fertility Index [%] = (No. of males impregnating a female/Total no. of males cohabited) x 100 :-[Measure of male's ability to produce sperm that can fertilise eggs]
Female Fertility Index [%] = (No. of pregnant females/No. of sperm-positive females) x 100 :-[Measure of female's ability to become pregnant]

Gestation Index [%] = (No. of females with live born pups/No. of pregnant females) x 100 :-[Measure of pregnancy that provides at least one live pup]

Offspring viability indices:

Survival Index [%] = (No. of live pups (at designated time)/No. of pups born) x 100

Pre-implantation mortality [%] = (No. of Corpora lutea - No. of Implantations/No. of Corpora lutea) x 100

Intrauterine mortality [%] = (No. of implantations - No. of liveborns/No. of implantations) x 100

Total mortality [%] = (No. of implantations - No. viable pups (PND4)/No. of implantations) x 100

% males = (No. of pups examined - No. of males/No. of pups examined) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

local effects

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no clinical signs related to treatment. All animals were clinically normal.
One male rat from the low dose group (10 mg/kg bw/day) was found dead. No specific cause of death was identified by necropsy, and the death was considered incidental and not test item related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No effects were noted on the body weight or body weight gain at 100, 30 and 10 mg/kg bw/day dose levels in males.
In females, occasional statistically significant increases in body weight group mean values were considered incidental to treatment.
There were no effects of the test-item on food consumption in males and females.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect of treatment on reproductive parameters.
There were no differences between the control and test item-treated groups with regard to reproductive ability, mating or gestation indices.
The mating indices were 100% in all groups, while female fertility indices were 83.3% in Control and High dose, 84.6 in the low dose and 91.7% in Mid dose animals. The gestation index was 100% in all groups.

Test item administration was considered to have no impact on the time to mating. The group mean pre-coital intervals were 2, 1, 2 and 2 days in the control, low, mid and high dose groups respectively. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 4 days of pairing (cohabitation).

There was no effect of treatment noted during gestation, parturition or the post-partal period.

The mean duration of pregnancy was similar in the control and test item treated groups and varied from 22.60 days (control and 100 mg/kg bw/day) to 22.73 days (10 mg/kg bw/day). All the parturitions were normal.

The number of corpora lutea and number of implantation sites were comparable to the control mean for all dose groups.

There were no significant differences or effects that could be ascribed to treatment on the pre-implantation loss, post-natal loss or total mortality values (litter mean and %) at any dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no toxicologically significant effects on organ weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Test item-related macroscopic findings were observed in the glandular mucosa of the stomach at a dose level of 100 mg/kg bw/day.

Red discoloration was present in the stomach mucosa in 12/12 males and 1/12 female rats, increased thickness of the stomach mucosa was present in 1/12 male, and a single ulcer of the gastric mucosa was present in 6/12 males and 1/12 female rats, were treatment-related effects observed at necropsy.

Other observations were considered to be incidental, or a common background observation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test item-related microscopic findings were noted in the glandular stomach mucosa at the High dose level of 100 mg/kg bw/day. The changes were characterized as red cytoplasmic glandular foreign material, mixed cellular diffuse inflammation of the glandular mucosa (with/without submucosal lymphoid follicle formation), and erosion or ulcer of the glandular mucosa (mainly fundic and pyloric). These lesions correlated with the gross findings recorded at necropsy.

The presence of red cytoplasmic glandular foreign material (with diffuse or multifocal distribution) was noted in 12/12 males and 1/12 female at the High dose level. Minimal to marked inflammation, with/without lymphoid follicle formation (predominantly mild severity), was present in 12/12 males and 1/12 female rats. Minimal to mild mucosal erosion was recorded in 3/12 males. Focal mild or moderate ulcers were present in 3/12 males and 1/12 female.

No test item-related microscopic changes were noted in the reproductive organs, at the High dose level of 100 mg/kg bw/day.

All other changes were incidental or considered to be common background findings in this strain sex and age of rat.

Effect levels (P0)

1

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

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic toxicity was observed. Treatment related effects in the high dose group were considered to reflect a local irritant effect of the test substance rather than systemic toxicity.

3

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The number of viable pups on PND0 and 4 at up to 100 mg/kg bw/day was comparable to the control values.

CLINICAL SIGNS (OFFSPRING)
Administration of the test item to the parental generation at up to 100 mg/kg bw/day did not cause mortality or any adverse effects considered to be treatment-related in the F1 generation. No abnormal behaviour of the pups was noted.

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

GROSS PATHOLOGY (OFFSPRING)
No test item-related macroscopic changes were seen in delivered pups.

OTHER FINDINGS (OFFSPRING)
The incidence of mortality during 4 postnatal days was negligible and was 2 of 138 (control), 3 of 170 (Low dose), 7 of 170 (Mid dose) and 3 of 161 (High dose). Slightly higher than control post natal mortality was noted in the mid dose groups (30 mg/kg bw/day), due to one dam with whole litter loss. This was not typical of the group or the higher dose and therefore considered incidental to treatment.

The survival indices on PND 0 and 4 were similar in all groups.

The sex ratios did not differ significantly between the control and treatment groups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

gross pathology

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a OECD guideline reproduction and developmental toxicity study, to GLP, parental (F0) rats (12/sex/group) were administered diammonium hexachloropalladate by gavage at 0 (corn oil), 10, 30 or 100 mg/kg bw/day. No adverse effects on reproductive parameters of parent males and females (F0 animals), or on developmental of offspring (FI generation), was observed at any dose, resulting in a NOAEL of 100 mg/kg bw/day for such effects.

Executive summary:

The potential of diammonium hexachloropalladate to adversely affect the fertility and reproductive parameters of rats was investigated in a guideline reproductive and developmental screening study (OECD TG 421), conducted according to GLP. The test material (in corn oil) 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 4 days mating period, through gestation and up to and including the day before necropsy (at least 4 days post-partum dosing). Three dose groups (10, 30 and 100 mg/kg bw/day) and a vehicle control group were used, each containing 12 males and 12 females (F0 animals).

 

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.

 

Test item-related macroscopic and microscopic findings were noted in parental animals in the glandular stomach mucosa at a dose level of 100 mg/kg bw/day (resulting in a NOAEL of 30 mg/kg bw/day for local effects). No systemic effects were seen in the parenteral animals (resulting in a NOAEL of 100 mg/kg bw/day for systemic effects). No test item-related microscopic changes were noted in the reproductive organs, at a dose level of 100 mg/kg bw/day. 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.