Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)

Endpoint summary

Description of key information

In an OECD Test Guideline 422 combined repeated dose 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 at doses of 0, 100, 300 or 1000 mg/kg bw/day. Control animals received vehicle only. 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 (Hansen, 2017).

 

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

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 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.

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.

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)

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.

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

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Overall, good-quality database which meets REACH Standard Information Requirements.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No data identified.

Additional information

No relevant human data were identified.

 

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. On this basis, the NOAEL for systemic toxicity was established as 300 mg/kg bw/day (Hansen, 2017).

 

According to REACH Annex VIII (EC 1907/2006), repeated dose toxicity studies only need to be conducted on one species taking into consideration the most appropriate route of administration regarding human exposure.

Justification for classification or non-classification

In a reliable repeated dose toxicity study (combined with a reproductive/developmental screening assay) involving gavage administration of dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol to rats for at least 28 days, no adverse systemic effects were seen at up to 300 mg/kg bw/day. As such, classification of this substance as STOT-RE is not required, according to EU CLP criteria (EC 1272/2008).