Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Part of a combined repeated dose study (OECD 422) with reproductive and developmental toxicity screening.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2016 - 6 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to GLP
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Part of a combined repeated dose study (OECD 422) with reproductive and developmental toxicity screening.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 November 2016 - 6 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to GLP
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
Justification for study design:
Not applicable
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16291C1AES.
- Expiration date of the lot/batch: 01 October 2019.
- Purity test date: 27 October 2016.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C; under inert gas (argon), in a tightly closed container in a dry place, protected from heat and direct sunlight.
- Stability under test conditions: Stable under prescribed storage conditions.
- Solubility and stability of the test substance in the solvent/vehicle: Satisfactory stability of suspensions of the test material in corn oil for 7 days has been previously demonstrated (LPT Report No. 33687).
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies.
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, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: 80 days.
- Weight at study initiation: Males: 404.3 g - 475.3 g / Females: 220.8 g - 279.9 g
- Fasting period before study: No.
- Housing: Housed singly, except during mating period.
- Diet (e.g. ad libitum): Certified commercial diet, provided ad libitum.
- Water (e.g. ad libitum): Tap water provided ad libitum.
- Acclimation period: 5 days.

DETAILS OF FOOD AND WATER QUALITY: Samples of both food and water are analysed periodically for quality; certificates of analysis were provided with the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C.
- Humidity (%): 55% +/- 15%.
- Air changes (per hr): 15-20 changes/hour/
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light (150 lux at approximately 1.5m room height).

IN-LIFE DATES: First application 20 December 2016 (aged 80 days). End of in-life period: 21 February 2017.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared once weekly and stored in a tightly closed container at room temperature (+10°C to +25°C) until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard non-aqueous vehicle for formulating suspensions.
- Concentration in vehicle: 20, 60 or 200 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg bw.
- Lot/batch no. (if required): Batch nos. 16260301 or 15422602, Caesar & Loretz GmbH, 40721 Hilden, Germany
- Purity: not specified.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of homogeneity and concentration conducted weekly on 3 triplicate samples (total 9 samples), immediately after preparation of the test item-vehicle formulations.
Duration of treatment / exposure:
Males: 2 weeks prior to mating (test days 15-29), during the mating period (maximum test days 30-43) and until test day 50.
Females: 2 weeks prior to mating (test days 15-29), during the mating period (maximum test days 30-43) and during the lactation period until test days 64-77 (corresponding to lactation days 13-15).
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 80 days (males and females); F1 animals killed at postnatal day 13
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
"low dose"
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
"intermediate dose"
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
"high dose"
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor based on available toxicological data and a 14-day dose range finding study (LPT Study No. 33687).

In the 14-day dose range finding study, Dihydrogen hexahydroxyplatinate / 2-amino-ethanol (1:2) concentrate was administered orally to male and female rats at dose levels of 500, 750 or 1000 mg/kg b.w./day for 2 weeks. At 750 mg/kg b.w./day a slight reduction in body weight was noted for the female animals (at maximum 6.1% below the value of the control group, statistically not significant). At 1000 mg/kg b.w./day slight reductions in body weight were noted for the male and female animals, statistically significant only for the female animals (6.7% below the value of the control group, p ≤ 0.05). Furthermore, a slight but statistically significant reduction in food consumption was noted for the male animals during the first test week (12.3% below the value of the control group, p ≤ 0.05).

No changes in behavior or the external appearance were noted. No findings were noted during the macroscopic inspection at necropsy. The organ weights of the kidneys, the liver, the testes and / or the ovaries revealed no test item-related differences between the control group and the test itemtreated groups. Based on the data obtained in this dose range finding study, dose levels of 100, 300 and 1000 mg Dihydrogen hexahydroxyplatinate / 2-amino-ethanol (1:2) concentrate/kg b.w./day were selected for the main study (LPT Study No. 33738).
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations checked included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before first administration and once weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum), and on days 4 and 13 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable (compound dosed by gavage)

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE: Drinking water consumption was monitored daily by visual appraisal throughout the study
Oestrous cyclicity (parental animals):
The oestrus cycle stage of each animal at necropsy was determined from the vaginal smears.
Sperm parameters (parental animals):
Parameters examined in male parental (Fo) animals: testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- 10 pups/litter following a randomisation scheme; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts (pups with body weight < 70% of mean litter weight), postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external gross abnormalities. The external reproductive genitals were examined for signs of altered development.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not examined

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not examined

OTHER: The thyroid of 1 male and 1 female pup from each litter was fixed. Thyroid weight were determined after fixation.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were euthanised by carbon dioxide inhalation and exsanguination on test day 48.
- Maternal animals: All surviving animals were euthanised by carbon dioxide inhalation and exsanguination on lactation day 14 or 16.

GROSS NECROPSY
- Gross necropsy consisted of weighing and examination of the organs listed in table 1.

HISTOPATHOLOGY
The tissues indicated in table 2 were prepared for microscopic examination.
Postmortem examinations (offspring):
SACRIFICE
- All pups were euthanised by carbon dioxide inhalation and exsanguination on post-natal day 13.
- Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
Statistics:
Parametrical data: Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data: The statistical evaluation of non-parametrical values was done using the FISHER or Chi-Squared test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01); or
Chi-Squared test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)
Reproductive indices:
- Female fertility index (%) = (Number of pregnant rats/Number of rats used) x 100

- Gestation index (%) = (Number of dams with live pups/Number of pregnant rats) x 100
Offspring viability indices:
- Birth index (%) = (Total nymber of pups born (alive + dead)/Number of implantation scars) x 100

- Live Birth Index (%) = (Number of pups alive on day 0/1 of lactation/Total number of pups born (alive + dead)) x 100

- Viability Index (%) = (Number of pups alive on day 4/Number of pups alive on day 0/1) x 100

- Post-implantation Loss (%) = ((Implantations - Living Foetuses)/Implantations) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males
Salivation (slight, incidental (low-dose) or slight-to-pronounced (intermediate- and high-dose)) reported in 1/10, 5/10 and 10/10 animals in the low-, intermediate- and high-dose animals, respectively. No other clinical signs observed in the low-dose group. Piloerection and haemorrhagic nose/snout, and breathing sounds were each observed in 1/10 intermediate-dose animals but considered incidental on the basis of incidence. Incidental haemorrhagic nose/snout was also reported in 1/10 high-dose animals. Breathing sounds were noted in 4/10, and slightly reduced motility in 10/10 high-dose animals.

Females
Slight to pronounced salivation was reported in in 2/10 intermediate-dose and 9/10 high-dose animals. In the high-dose group, slightly reduced motility was reported in 10/10 animals, and breathing sounds were noted in 1/10 animals. No clinical signs were observed in low-dose females.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No premature death was noted in the control group or in the treatment groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
A slightly-reduced body weight compared to controls was recorded from test day 29 (4.6% below controls, not statistically significant) until sacrifice (test day 51, 8.6% below controls, not statistically significant). Body weight gain for high-dose males was "clearly below" the control group for the whole treatment period (test days 15-51; 3.8% gain for high-dose animals vs. 13.2% in controls). At autopsy, high-dose males had a body weight 9.4% below controls (not statistically significant).

Females
Slight, but statistically not significant, reductions in body weight were reported in high-dose females at the end of gestation and during lactation (6.5% below controls on gestation day 20, 5.8% below controls on lactation day 1 and 5.0% below controls on lactation day 13). At autopsy, high-dose females had a body weight 2.4% below controls (not statistically significant).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males
A slight, statistically not significant, reduction in food intake was noted during the first 2 weeks of dosing in the high-dose group (9.0% and 6.0% below controls).

Females
A statistically not significant reduction in food intake was noted in high-dose animals during the first week of dosing (8.1% below controls). A reduction was also seen in the high-dose animals throughout the gestation period (10.5% below controls in the first week (statistically significant), 7.4% below controls in the second week (not statistically significant) and 10.0% below controls in the third week (statistically significant). A slight but statistically significant reduction in food consumption was also noted in intermediate-dose animals during the third week of gestation (8.1% below controls). This was not considered by the study authors as test item-related.
Food efficiency:
not examined
Description (incidence and severity):
No test item-related changes in the consumption of drinking water was noted by visual appraisal for any treated rats.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the haematological parameters of the male and female animals in any of the treatment groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item related influence was noted for the examined plasma levels of the biochemical parameters in any of the treatment groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No observations of abnormal behaviour or test item-related differences in body temperature or the hind-leg splay in comparison to the control group were noted for the male and female animals of all treatment groups. No test item-related influence on the fore- and hindlimb grip strength or in spontaneous motility was noted for the male and female animals in any of the treatment groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males
Test item-related stomach lesions were reported in high-dose animals. Specifically, 3/5 animals examined showed erosions in the glandular mucosa, haemorrhage, inflammation, pigment depositon and ulcers in 1/5, and inflammation of the submucosa of the forestomach in 1/5. However, these findings could be related to the deposition of the test item and were not considered as an adverse effect of the test item.

Females
No test item-related microscopic findings were reported in the 5 high-dose females subjected to examination.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test item-related differences in thyroid (T4) hormone levels were noted between the male animals of the control group and the male animals of the treatment groups. Likewise, there were no test-item related differences between T4 levels in control or treated pups. Blood samples taken from treated dams at scheduled sacrifice were not analysed for T4 level.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test item-related influence was noted.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no test item-related influence noted on the fertility index, gestation index, pre-coital time or gestation length of treated animals.
There were no test item-related microscopic changes in the reproductive organs of the male animals. The histopathological examination that was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure) did not reveal any test item-related effects.

No test item-related microscopic changes could be observed in the reproductive organs for group 4 females that were examined microscopically. The mammary glands of females observed showed prominent mammary development.
Dose descriptor:
NOAEL
Remarks:
Fertility and reproductive parameters
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive effects seen at highest tested dose
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No test item-related differences were noted between the viability index of the pups from the dams of the control group and the pups from the treated dams.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the treatment groups on lactations days 1, 4 and 13.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related differences in the weight of the thyroid glands of the male and female pups in the controls groups compared to the treatment groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross abnormalities (e.g. malformations) were noted during the macroscopic external examination of the control pups and the pups from the treated dams.
Histopathological findings:
no effects observed
Description (incidence and severity):
The microscopic examination of the thyroids of the pups revealed no changes in the pups of the control group and the pups of the treatment groups.
Other effects:
no effects observed
Description (incidence and severity):
No test item-related differences were noted for the mean number of implantation sites, pups born (alive and dead) and live born pups between the control group and the treatment groups. Correlating the reproductive indices birth index, the live birth index and the percentage of post implantation loss were not influenced by the test item at any tested dose level.

No test item-related differences were noted for the thyroid (T4) hormone levels of the 13 day old pups.

No test item-related difference was noted for the ano-genital distance of the male and the female pups between the control group and the treatment groups. No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Remarks:
adverse effects on prenatal development (conceptus to birth)
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen at highest tested dose
Dose descriptor:
NOAEL
Remarks:
adverse effects on postnatal development (pup)
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen at highest tested dose
Critical effects observed:
not specified
Reproductive effects observed:
no
Conclusions:
In an OECD Test Guideline 422 combined repeated dose and reproductive/developmental toxicity screening study in rats, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol was administered by gavage for at least 28 days at doses of 0, 100, 300 or 1000 mg/kg bw/day. No effects on any measured reproductive or fertility parameters were observed. The 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, CD rats (10/sex/group) were orally administered dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol by stomach tube (gavage, in corn oil) at doses of 0, 100, 300 or 1000 mg/kg bw/day. Males were dosed for at least 28 days (14 days pre-mating, as well as during the mating and post-mating periods). Females were dosed for 14 days pre-mating, through mating, gestation (around 22 days) and up to post-natal day 13 (50 -63 days in total). Control animals received vehicle only.

There were no test item-related adverse effects on the measured reproductive or fertility parameters, on the pre-natal (pre- and post-implantation loss, number of pups born, number of stillbirths, birth and live birth indices) or the post-natal (pup body weight, survival index, the endocrine/sexual development (T4 levels, ano-genital distance, male nipples counting), gross abnormalities) developmental of the pups. Consequently, the NOAELs for reproductive toxicity, pre-natal and post-natal development were all set at 1000 mg/kg bw/day, the highest dose tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)
EC Number:
268-717-3
EC Name:
Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)
Cas Number:
68133-90-4
Molecular formula:
C2H7NO.1/2H6O6Pt.H
IUPAC Name:
dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2)
- Substance type: No data
- Physical state: yellow/orange coloured liquid
- Analytical purity: No data
- Impurities (identity and concentrations in pmm): Ag (<0.5), Al (12), As (<10), Au (<10), Bi (<5), Ca (36), Cr (<2), Cu (<5), Fe (34.0), Ir (<3), K (<5), Mg (17.0), Mn (<1), Mo (<5), Na (3096), Ni (<5), Pd (<5), Rh (40), Ru (<7), Sb, (<15), Si (172), Sn (<5), Zn (<0.5), Cl- (1096)
- Composition of test material, percentage of components: Platinum content 17.15% (w/w)
- Isomers composition: Not applicable
- Purity test date: 26 May 2015
- Lot/batch No.: 15135C1AES
- Expiration date of the lot/batch: 25 May 2018
- Stability under test conditions: No data
- Storage condition of test material: At +10°C to +25°C, kept in a tightly closed container and stored in a dry place, protected from light.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16291C1AES.
- Expiration date of the lot/batch: 01 October 2019.
- Purity test date: 27 October 2016.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C; under inert gas (argon), in a tightly closed container in a dry place, protected from heat and direct sunlight.
- Stability under test conditions: Stable under prescribed storage conditions.
- Solubility and stability of the test substance in the solvent/vehicle: Satisfactory stability of suspensions of the test material in corn oil for 7 days has been previously demonstrated (LPT Report No. 33687).

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies.
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, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: 80 days.
- Weight at study initiation: Males: 404.3 g - 475.3 g / Females: 220.8 g - 279.9 g
- Fasting period before study: No.
- Housing: Housed singly, except during mating period.
- Diet (e.g. ad libitum): Certified commercial diet, provided ad libitum.
- Water (e.g. ad libitum): Tap water provided ad libitum.
- Acclimation period: 5 days.

DETAILS OF FOOD AND WATER QUALITY: Samples of both food and water are analysed periodically for quality; certificates of analysis were provided with the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C.
- Humidity (%): 55% +/- 15%.
- Air changes (per hr): 15-20 changes/hour/
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light (150 lux at approximately 1.5m room height).

IN-LIFE DATES: First application 20 December 2016 (aged 80 days). End of in-life period: 21 February 2017.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared once weekly and stored in a tightly closed container at room temperature (+10°C to +25°C) until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Standard non-aqueous vehicle for formulating suspensions.
- Concentration in vehicle: 20, 60 or 200 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg bw.
- Lot/batch no. (if required): Batch nos. 16260301 or 15422602, Caesar & Loretz GmbH, 40721 Hilden, Germany
- Purity: not specified.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of homogeneity and concentration conducted weekly on 3 triplicate samples (total 9 samples), immediately after preparation of the test item-vehicle formulations.
Duration of treatment / exposure:
Males: 2 weeks prior to mating (test days 15-29), during the mating period (maximum test days 30-43) and until test day 50.
Females: 2 weeks prior to mating (test days 15-29), during the mating period (maximum test days 30-43) and during the lactation period until test days 64-77 (corresponding to lactation days 13-15).
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
"low dose"
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
"intermediate dose"
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
"high dose"
No. of animals per sex per dose:
10.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected by the Sponsor based on available toxicological data and a 14-day dose range finding study (LPT Study No. 33687).

In the 14-day dose range finding study, Dihydrogen hexahydroxyplatinate / 2-amino-ethanol (1:2) concentrate was administered orally to male and female rats at dose levels of 500, 750 or 1000 mg/kg b.w./day for 2 weeks. At 750 mg/kg b.w./day a slight reduction in body weight was noted for the female animals (at maximum 6.1% below the value of the control group, statistically not significant). At 1000 mg/kg b.w./day slight reductions in body weight were noted for the male and female animals, statistically significant only for the female animals (6.7% below the value of the control group, p ≤ 0.05). Furthermore, a slight but statistically significant reduction in food consumption was noted for the male animals during the first test week (12.3% below the value of the control group, p ≤ 0.05).

No changes in behavior or the external appearance were noted. No findings were noted during the macroscopic inspection at necropsy. The organ weights of the kidneys, the liver, the testes and / or the ovaries revealed no test item-related differences between the control group and the test item-treated groups. Based on the data obtained in this dose range finding study, dose levels of 100, 300 and 1000 mg Dihydrogen hexahydroxyplatinate / 2-amino-ethanol (1:2) concentrate/kg b.w./day were selected for the main study (LPT Study No. 33738).
Positive control:
Not applicable.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations checked included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before first administration and once weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum), and on days 4 and 13 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable (compound dosed by gavage)

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE: Drinking water consumption was monitored daily by visual appraisal throughout the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period (test day 29)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group, selected randomly
- Parameters checked in tables 1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period (test day 29)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex/group, selected randomly
- Parameters checked in table 3 were examined.

THYROID HORMONE (T4) DETERMINATION
- Time schedule for collection of blood: At least 2 surplus pups/litter, from all litters, on post-natal day (PND) 4; at least 2 pups/litter, from all litters, on PND 13; all evaluated dams and all adult males at scheduled sacrifice

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Two hours after dosing. 5 males/group (randomly selected) were evaluated on test day 45; 5 females/group were evaluated between lactation days 13 and 15.
- Dose groups that were examined: All
- Battery of functions tested: See table 4

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes (see table 6)
Statistics:
Parametrical data: Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data: The statistical evaluation of non-parametrical values was done using the FISHER or Chi-Squared test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01); or
Chi-Squared test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males
Salivation (slight, incidental (low-dose) or slight-to-pronounced (intermediate- and high-dose)) reported in 1/10, 5/10 and 10/10 animals in the low-, intermediate- and high-dose animals, respectively.
No other clinical signs observed in the low-dose group. Piloerection and haemorrhagic nose/snout, and breathing sounds were each observed in 1/10 intermediate-dose animals but considered incidental on the basis of incidence.
Incidental haemorrhagic nose/snout was also reported in 1/10 high-dose animals. Breathing sounds were noted in 4/10, and slightly reduced motility in 10/10 high-dose animals.

Females
Slight to pronounced salivation was reported in in 2/10 intermediate-dose and 9/10 high-dose animals. In the high-dose group, slightly reduced motility was reported in 10/10 animals, and breathing sounds were noted in 1/10 animals.
No clinical signs were observed in low-dose females.
Mortality:
no mortality observed
Description (incidence):
No premature death was noted in the control group or in the treatment groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
A slightly-reduced body weight compared to controls was recorded from test day 29 (4.6% below controls, not statistically significant) until sacrifice (test day 51, 8.6% below controls, not statistically significant). Body weight gain for high-dose males was "clearly below" the control group for the whole treatment period (test days 15-51; 3.8% gain for high-dose animals vs. 13.2% in controls). At autopsy, high-dose males had a body weight 9.4% below controls (not statistically significant).

Females
Slight, but statistically not significant, reductions in body weight were reported in high-dose females at the end of gestation and during lactation (6.5% below controls on gestation day 20, 5.8% below controls on lactation day 1 and 5.0% below controls on lactation day 13). At autopsy, high-dose females had a body weight 2.4% below controls (not statistically significant).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males
A slight, statistically not significant, reduction in food intake was noted during the first 2 weeks of dosing in the high-dose group (9.0% and 6.0% below controls).

Females
A statistically not significant reduction in food intake was noted in high-dose animals during the first week of dosing (8.1% below controls). A reduction was also seen in the high-dose animals throughout the gestation period (10.5% below controls in the first week (statistically significant), 7.4% below controls in the second week (not statistically significant) and 10.0% below controls in the third week (statistically significant). A slight but statistically significant reduction in food consumption was also noted in intermediate-dose animals during the third week of gestation (8.1% below controls). This was not considered by the study authors as test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in the consumption of drinking water was noted by visual appraisal for any treated rats.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on the haematological parameters of the male and female animals in any of the treatment groups.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item related influence was noted for the examined plasma levels of the biochemical parameters in any of the treatment groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No observations of abnormal behaviour or test item-related differences in body temperature or the hind-leg splay in comparison to the control group were noted for the male and female animals of all treatment groups. No test item-related influence on the fore- and hindlimb grip strength or in spontaneous motility was noted for the male and female animals in any of the treatment groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related influence was noted for the relative or absolute organ weights of the male or female animals in any of the test item-treated groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
5/10 high-dose animals showed ulcers or thickened and/or discoloured cardiac stomach. These observations were confirmed by microscopic examination in 2/5 animals (the stomachs of the other 3/5 affected animals were not examined microscopically). However, these findings could be related to the deposition of the test item and were not considered as an adverse effect of the test item.

Females
No test item-related macroscopic changes reported in any treated females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males
Test item-related stomach lesions were reported in high-dose animals. Specifically, 3/5 animals examined showed erosions in the glandular mucosa, haemorrhage, inflammation, pigment depositon and ulcers in 1/5, and inflammation of the submucosa of the forestomach in 1/5. However, these findings could be related to the deposition of the test item and were not considered as an adverse effect of the test item.
There were no test item-related microscopic changes in the reproductive organs of the male animals. The histopathological examination that was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure) did not reveal any test item-related effects.

Females
No test item-related microscopic findings were reported in the 5 high-dose females subjected to examination.
No test item-related microscopic changes could be observed in the reproductive organs for group 4 females that were examined microscopically. The mammary glands of females observed showed prominent mammary development.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test item-related differences in thyroid (T4) hormone levels were noted between the male animals of the control group and the male animals of the treatment groups. Likewise, there were no test-item related differences between T4 levels in control or treated pups. Blood samples taken from treated dams at scheduled sacrifice were not analysed for T4 level.

Effect levels

Dose descriptor:
NOAEL
Remarks:
general (systemic) toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In an OECD Test Guideline 422 combined repeated dose toxicity and reproduction/developmental toxicity screening study in rats, conducted to GLP, dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol, was administered by gavage for at least 28 days (males) or 50-63 days (females) at doses of 0, 100, 300 or 1000 mg/kg bw/day. Treatment-related adverse effects were observed in the high dose animals (including reduced motility, decreased food consumption and body weight). The NOAEL for systemic toxicity was established as 300 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, CD rats (10/sex/group) were orally administered dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol by stomach tube (gavage, in corn oil) at doses of 0, 100, 300 or 1000 mg/kg bw/day. Males were dosed for at least 28 days (14 days pre-mating, as well as during the mating and post-mating periods). Females were dosed for 14 days pre-mating, through mating, gestation (around 22 days) and up to post-natal day 13 (50 -63 days in total). Control animals received vehicle only.

In the high-dose group, animals demonstrated clinical signs of toxicity including salivation, breathing sounds and reduced motility, as well as decreased food consumption and body weight. Gross lesions to the stomach were also observed and confirmed by further microscopic examination, but might have arisen due to deposition of the test item and were not considered as an adverse effect of the test item.

The NOAEL for systemic toxicity was established as 300 mg/kg bw/day.