Diammonium hexachloropalladate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 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

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

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.

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.

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.

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

Examinations

Maternal 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, 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
- 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all]
- Soft tissue examinations: No data
- Skeletal examinations: No data
- Head examinations: No data

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.

Indices:

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

Historical control data:

No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: local effects

Details on maternal toxic effects:
Test item-related macroscopic and microscopic findings were noted in the glandular stomach mucosa at the top dose (100 mg/kg bw/day). The changes were characterized as red cytoplasmic glandular foreign material, mixed cellular diffuse inflammation of glandular mucosa with/without submucosal lymphoid follicle formation, erosion or ulcer of the glandular mucosa (mainly fundic and pyloric). These lesions were in correlation with macroscopic findings seen at necropsy, and considered to reflect a local effect.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Embryotoxicity, teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

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 parental (F0) animals, or on development 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 development of rats was investigated in a guideline reproductive and developmental screening study (OECD TG421), 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.

 

Parental (F0) animals were observed for clinical signs of toxicity throughout the study, with body weights and food consumption monitored. At necropsy, animals were subjected to external and internal macroscopic examinations for any abnormalities or pathological changes. Special attention was paid to the reproductive organs. Pups were carefully examined for gross abnormalities at necropsy (on postnatal day 4).

 

Macroscopic and microscopic findings were noted in parental animals in the glandular stomach mucosa at a dose level of 100 mg/kg bw/day, reflecting a treatment-related local effect. No systemic toxicity was observed in the F0 animals. There was no developmental toxicity effect at any dose level. Consequently, the NOAEL for developmental toxicity was 100 mg/kg bw/day, the highest dose tested.