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Description of key information

In the Lumière Pink S.M. 8135N: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study in the Wistar Rat by Oral Gavage Administration it has been concluded that, despite the histopathological findings observed in kidneys at 100 mg/kg (tubular basophilia), considering that they were of minimal to slight severity, partly consistent with a compensatory reparation and taking into account that there is no clear evidence that there is an affectation at functional level given the absence of biochemical effects at this dose, NOAEL can be considered as the dose of 100 mg/kg/day within the confines of this study.

  

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2017 - 21 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 021340
- Expiration date of the lot/batch: July 2022
- Purity test date: 25 August 2003
- Appearance: Red powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at ambient conditions in the dark (away from direct light).
- Stability under test conditions: 8 days
- Solubility and stability of the test substance in the solvent/vehicle: Yes



Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and strain selected to maintain consistentcy with previous toxicological study
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males: 10 weeks
Females: 10 weeks

- Weight at study initiation: Males: 256-359 g
Females: 186-248 g

- Housing: Cages with standard, granulated, S8-15 sawdust bedding.
Premating period (four animals/cage) Makrolon type-IV cages
Mating period (one male and one female/cage) Makrolon type-III cages
Postmating, gestation and lactation periods (individual) Makrolon type-III

- Diet: ad libitum , standard commercial diet
- Water: ad libitum, tap water
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): between 30 and 70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 fluorescent light/12 hours dark
Route of administration:
oral: gavage
Details on route of administration:
Oral by gastric gavage - as oral ingestion is a possible route for human exposure to the test item.
For each dose group the order of administration was all males first and then all females. Each day of treatment the starting order was alternated between males and females. It was stated in the raw data.
Formulations were maintained under agitation before their administration for 3 ¾ to 24 hours (until the end of administration). The start time of agitation was recorded in the raw data..
Vehicle:
arachis oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
High Concentration Formulation (GROUP 4)
The necessary amount of test item was weighed in a disposable flask. 70-80% of vehicle (Arachis oil) required to reach the final formulation volume was poured into an empty disposable flask and the test item was added.
The content of the flask was mixed using an Ultra-Turrax or a homogenizer (depending on the total volume of formulation prepared) until complete homogenization.
The suspension was then transferred to a volumetric vessel. A small amount of vehicle was used to remove any formulation remaining in the disposable flask and the mixture was poured into the vessel. Vehicle was then added to the volumetric vessel to make up to volume.
Finally, the suspension was transferred to an appropriate container and submitted to magnetic stirring for 10 minutes before sampling for dilution.
Dilution Preparation: Intermediate and Low concentration Formulations (Groups 2 and 3)
It was prepared by diluting the high formulation and submitted to magnetic stirring for 10 minutes before aliquoting.
The density was taken the first day of preparation of formulation. Formulations were maintained under agitation for 3 ¾ to 24 hours until the end of the administration, assuring that formulations were homogenous during their administration.

- VEHICLE
- Concentration in vehicle: 20 mg/mL to 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no. (if required): KMO9422
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify correct preparation.
Control (vehicle) formulations were analyzed to confirm the absence of test item or other substances at the retention time of the test item.
The test item was used as analytical standard.
Results were expected to be within ±20% of nominal value and the coefficient of variation (CV) should be ≤10%.
Duration of treatment / exposure:
5-8 weeks
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: It was considered a suitable dose level range, based on the preliminary results obtained in the previous non-GLP study PQ36XP 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at in Envigo CRS, S.A.U.:
- The high dose was selected as no toxicity was observed in the preliminary study at 1000 mg/kg/day and considering it as a limit dose to be tested.
- Intermediate and low dose levels were selected considering approximately a 3-fold interval between doses.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twicw daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
During mating: Once a week for F0 males and F0 females
During post-mating: Once a week for F0 males
During gestation: Once a week for F0 females
During lactation: Once a week for F0 females
During Littering phase: Observed 24 hours after the considered birth and then daily for evidence of illhealth
or reaction to maternal treatment

BODY WEIGHT: Yes
- Time schedule for examinations:
During mating: Weekly for F0 males and F0 females
During post-mating: Weekly for F0 males
During gestation: On days: 0, 7, 14, 20 for F0 females
During lactation: On days: 1, 4 and 13 for F0 females
During Littering phase: Days 1, 4, 7 and 13 of age for individual offspring

Sensory Reactivity and Grip Strength: Yes.
- Time schedule for examinations: The observaions waere performed in the week 5 (before dosing) for
males and on the days 7 of lactation (before dosing) for females.

Motor activity: Yes.
- Time schedule for examinations: The observaions waere performed in the week 5 (before dosing) for
males and on the days 7 (before dosing) for females.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: At termination
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked in table [2] were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3 and 4)

HISTOPATHOLOGY: Yes (see table 3 and 4)
Statistics:
All statistical analyses were carried out separately for males and females. The following comparisons were performed: Group 1 vs. 2, 3 and 4.
For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity is not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there is evidence against a monotonic dose-response when Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test is still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test, unless there is evidence against a monotonic dose response when Steel's test was performed instead.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Female 96, administered at 1000 mg/kg/day, was sacrificed on lactation day 2 because it showed no maternal care. Consequently, the whole litter showed signs of hypothermia and no milk present in the stomach and as observed during the veterinary inspection, decreased or no motor activity was observed. This litter was sacrificed for welfare reasons. No findings were observed in the macroscopic examination. The major factor contributing to their death could not be determined in the absence of any histopathological finding. Maternal neglect in this case could be considered occasional and not treatment related, given the incidence observed in the study and as no test item related effect was observed in this animal during the study.
Pink colored feces (due to test item color) were observed from day 3 in males and 2 in females until the end of the study. Pink feces were evident at all doses of the test item.
Salivation appeared in males and females administered at 1000 mg/kg/day from day 4 of treatment and until the end of the study it usually occurred approximately within ½ hour of dosing. Incidence was higher in males than in females.
No other signs considered to be related to treatment of Lumière Pink S.M. 8135N were observed during the study.
Mortality:
no mortality observed
Description (incidence):
Please refer to the above-section
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Treatment with Lumière Pink S.M. 8135N had no relevant effect on body weight.
From treatment until the end of the study, males receiving 1000 mg/kg/day had slightly lower mean body weights than control animals. However, the lower weight cannot be considered relevant due to its magnitude, to the fact that body weight gain was similar to controls and because the same effect was not observed in females.
Differences observed in males at the end of post-mating period are related with fasting prior to necropsy.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Treatment with Lumière Pink S.M. 8135N has no effects on food consumption measurements during the whole study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry after 6-7 weeks of treatment in males and on day 16 of lactation revealed statistically higher than control creatinine levels at 300 and 1000 mg/kg/day in males and lower than control bile acids in males at 1000 mg/kg/day. A trend to higher urea mean values in the test item administered groups was observed in males and females. Females receiving 1000 mg/kg/day had statistically higher urea values than controls.
Statistically significant higher potassium, chloride and calcium means were recorded in males at 1000 mg/kg/day when compared to control. The higher chloride mean value was also significant compared to control at 300 and 1000 mg/kg/day in males.
As a general rule, increased albumin and albumin/globulin ratio mean values at 300 and 1000 mg/kg/day were recorded in both sexes (they were statistically significant at1000 mg/kg/day for albumin and for albumin/globulin ratio at 300 and 1000 mg/kg/day).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity conducted during week 5 in males and between days 7-9 of lactation in females revealed no findings which were considered treatment related.
Grip strength recordings revealed a significant dose-related increase in mean hind limb values in females. However there was no evidence observed in clinical signs that can corroborate this finding and it was not observed in males.
The motor activity assessment conducted during week 5 in males and between days 7-9 of lactation in females revealed no treatment related effects in males and females at all dose levels.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and body-weight-adjusted adrenal weights in males and kidney weights in females were higher than control in all test item administered groups (100, 300 and 1000 mg/kg/day) and attained significant differences when compared to control. Differences were dose related.
Statistically significant differences were observed at 300 mg/kg/day and 1000 mg/kg/day in ovaries.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes that were considered related to treatment with the test item were seen in:
- Kidneys (renal cortex): minimal to moderate tubular basophilia in both genders in all test item administered groups. Tubules of the most affected males showed karyomegaly and were accompanied by minimal, multifocal, interstitial mononuclear cell infiltrates.
- Thymus of both sexes at 1000 mg/kg/day: minimal involution/atrophy or minimally increased cortical apoptosis was seen and cortical hypertrophy was also observed in the affected females.
- Adrenal glands of females at 1000 mg/kg/day: bilateral, minimal to slight diffuse cortical hypertrophy.
All other histological findings were considered to be incidental and unrelated to the test item.
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted in males treated at 1000 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
In conclusion, the effects of oral (gavage) administration of Lumière Pink S.M. 8135N to Wistar rats receiving 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:
Systemic toxicity:
- Despite the histopathological findings observed in kidneys at 100 mg/kg (tubular basophilia), considering that they were of minimal to slight severity, partly consistent with a compensatory reparation and taking into account that there is no clear evidence that there is an affectation at functional level given the absence of biochemical effects at this dose, NOAEL can be considered as the dose of 100 mg/kg/day within the confines of this study.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline study Klimisch 1
System:
urinary
Organ:
kidney

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

Additional information

Justification for classification or non-classification

Based on the results of the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Study in the Wistar Rat by Oral Gavage Administration (Envigo, 2018, PW94LF) the substance is classified as a STOT RE Cat 2 (kidneys).