Ethanaminium, N-[4-[[4-(diethylamino)phenyl][4-(ethylamino)-1-naphthalenyl]methylene]-2,5-cyclohexadien-1-ylidene]-N-ethyl-, molybdatetungstatephosphate

Endpoint summary

Description of key information

In a combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening with Pigment Blue 1 the No-Observed-Effect Level (NOEL) for general toxicity was considered to be 100 mg/kg bw/day, taking into account that there was mortality, lower body weighs and histopathological findings in kidneys (tubular basophilia) were recorded at 300 and 1000 mg/kg/day with respect to Control. Based on these findings and on the fact that litter loss does not seem to be dose-related, the No Observed Adverse Effect Level (NOAEL) could be established at 100 mg/kg/day, as the increased incidence of adrenal cortical hypertrophy with respect to controls is considered mainly a response to stress.

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:

22 August 2017 - 21 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

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: 981108
- Purity:Preparation containing ≥90% UVCB (treat as 100%). No correction factor for purity was applied
- Expiration date of the lot/batch: July 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at ambient conditions in the dark (away from direct light)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 255-323 g, Females: 167-218 g
- Housing: Optimum hygienic conditions behind a barrier system
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): Relative humidity between 30 and 70%.
- Air changes (per hr): airconditioned with a minimum of 15-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/12 hours dark.

Route of administration:

oral: gavage

Details on route of administration:

Oral, by gastric gavage. 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.

Vehicle:

arachis oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Compatible with previous toxicity study.
- Concentration in vehicle: 20-200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no.: 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 their correct preparation. Control (vehicle) formulations were taken and 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.

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 non-GLP study HJ88HL 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at 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: Twice 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 ill-health 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, 9 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 were performed in the week 5 (before dosing) for males and on the days 7-9 of lactation (before dosing) for females.

Motor activity: Yes.
- Time schedule for examinations: The observaions were performed in the week 5 (before dosing) for males and on the days 7-9 (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
- 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, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, one female was sacrificed on gestation day 21. Piloerection, reddish eye discharge, pasty feces, hunched posture and paleness of the whole body skin were recorded on day 21. During partum, 17 pups were delivered, showing 6 of them signs of cannibalization and 3 pups were missing. None of these necropsied pups showed milk in the stomach. This female did not show any macroscopic findings at necropsy.
One Female at 1000 mg/kg/day was killed on lactation day 1 as all her pups were found dead showing no milk in the stomach. One of these pups showed signs of cannibalization in right hindlimb. Before her sacrifice, the female showed decreased activity, reduced body tone, hunched posture, pallor and low body temperature. On gestation day 14, reddish discharge in vagina was observed in this female. No test-item-related effects were observed in the macroscopic examination in this female.

One Female administered at 1000 mg/kg/day did not take care of her pups and some of them were found dead between days 21 of gestation and 2 of lactation (showing no milk in the stomach). During the pup clinical session done on lactation day 1, these pups were found scattered and cold. This female cannibalized the rest of pups on lactation day 2 and consequently, this female was killed this day. No clinical signs were recorded for this female and no macroscopic findings were observed at necropsy.

On gestation day 23, one female dosed at 1000 mg/kg/day was found dead. No other clinical sign other than salivation was observed in this female during the study. Severe autolysis was observed during necropsy but there were no other findings. Five autolytic fetuses were found in the right uterine horn and eight in the left one.
One Female at 300 mg/kg/day was sacrificed for welfare reasons on gestation day 23, showing signs of dystocia (decreased activity, piloerection, reddish discharge in nose and paleness of the whole body skin). Dilated renal pelvis in left kidney and pale right kidney were recorded during necropsy. When uterine horns were observed internally, six male pups and six female pups were observed showing all of them signs of autolysis and edema.

On gestation day 23, one female at 300 mg/kg/day was found dead. No other clinical signs other than salivation were observed during the study and no macroscopic findings were recorded during necropsy. Six male pups and five female pups were found in the uterine horns with slight signs of autolysis and edema. No other macroscopic alterations were observed in these pups.

One Female administered at 300 mg/kg/day had cannibalized its pups on lactation day 2. On lactation day 1, its pups were found to be cold to touch and little or no milk was present in their stomach. Consequently, this female was sacrificed on lactation day 4. No macropathology findings were observed at necropsy.

At 100 mg/kg/day, one female cannibalized its litter on lactation day 3 and female no. 82 did not show maternal care. These females were consequently killed on lactation days 3 and 2, respectively. This Female showed hunched posture on lactation days 1 and 2 and its pups were scattered with no or little milk in the stomach and they were cold to touch. No macroscopic findings were observed in the females at necropsy and no abnormal findings other than no milk present in the stomach were observed in the sacrificed pups from this litter on day 2 of lactation.

One Female at 100 mg/kg/day did not give birth on day 26 of gestation and was subsequently killed. This female was found to be not pregnant (failed to litter). Blue feces (due to the test item color) were observed in males and females from day 2 until the end of the study. Blue feces were evident in all test-item-administered groups consequent to the test item color. Salivation appeared in males and females administered at 1000 mg/kg/day from day 11 of treatment and from mating at 100 and 300 mg/kg/day until the end of the study. This clinical sign tended to occur approximately within ½ hour after dosing. Incidence was similar in males and females. Salivation pre- or post-dosing is related with taste aversion in gavage dosing.

No other signs considered related to treatment with Lumière Blue PTM 0154N were observed
during the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

Please refer to the description under Clinical signs.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment of Lumière Blue PTM 0154N had no relevant effect on body weight. From mating until the end of the study, mean body weights for males at 300 and 1000 mg/kg/day were lower than in the Control group. In females, this was also observed at 1000 mg/kg/day, mainly during lactation. Statistically significant differences were observed from mating until post-mating day 15 in males and on lactation days 4 and 13 in females. Given the magnitude of the change (no more than 9% with respect to Control) and the body weight profile, it cannot be considered relevant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment with Lumière Blue PTM 0154N resulted in an increase in food consumption in males with respect to Control (with the exception of treatment period days 1-8 and postmating period days 15-21). These differences were mainly evident from post-mating start onwards.
In females, during lactation, lower food consumption values with respect to Control were observed at 1000 mg/kg/day.
Thus the trend in food consumption is not considered toxicologically relevant in the absence of a similar trend in both sexes and taking into account the magnitude of the differences.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No changes of toxicological relevance were observed in hematology or coagulation investigations after 5 weeks of treatment in males and on days 14-16 of lactation. A dose-related trend to lower platelet values in males and females was observed with respect to Control. In females, statistically significant differences were observed at 1000 mg/kg/day.
Statistically significant differences observed with respect to the controls (neutrophils, monocytes and large unstained cells in all test-item-administered males and in MCHC and RDW in females at 1000 mg/kg/day) were minor, lacked dose-effect relationship, were not present in both sexes and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry after 5 weeks of treatment in males and on day 14-16 of lactation in females revealed significantly higher mean alanine transferase and urea values at 1000 mg/kg/day in females compared to Control. A trend towards a dose-related increase in mean creatine kinase values was also observed in females, being these differences statistically significant at 300 and 1000 mg/kg/day. These observations were not present in males.
In males, statistically significant differences when compared to Control were recorded in mean HDL, calcium and total protein (lower values with respect to Control), in LDL and chloride (higher values with respect to Control) at 1000 mg/kg/day and in triglycerides at 100 mg/kg/day (higher values with respect to Control). In females, statistically significant differences were observed in potassium and calcium at 1000 mg/kg/day (higher values with respect to Control). These differences were not considered relevant given their magnitude and the fact that they are not consistent among sexes.
Statistically significant differences from controls observed in electrophoresis in the test item administered groups were considered minimal and occasional and were therefore attributed to normal biological variation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Adjusted liver and adrenal weights were higher in females than in Control mainly at 300 and 1000 mg/kg/day; the differences attained statistical significance with respect to control at 1000 mg/kg/day. Differences were dose-related. In males, they were also observed in adjusted liver weight, but not attaining statistical significance.
Adjusted thymus weight in females was lower than in Control in all test-item-administered groups (100, 300 and 1000 mg/kg/day), attaining statistical significance with respect to Control at 300 and 1000 mg/kg/day. Differences were dose-related.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength assessment conducted during week 5 in males and between days 7-9 of lactation in females revealed no findings.

The motor activity assessment conducted during week 5 in males and between days 7-9 of lactation in females showed no dose-related statistically significant differences in the test item administered males at 30 and 40 minutes and in the whole observation period. These differences were no observed in females. These differences were devoid of any toxicological significance and were attributed to the variability of this parameter more than to treatment
with Lumière Blue PTM 0154N.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes considered related to treatment with the test item were seen in the thymus and spleen of both sexes, and in the kidneys and adrenal glands of females:
- Minimal, bilateral, diffuse tubular basophilia was observed in the renal cortices of most examined females given the test item at 1000 mg/kg and in one female receiving 300 mg/kg. One control male showed only minimal isolated foci of basophilic tubules, within normal background levels.
- In adrenal glands, high incidence of bilateral diffuse cortical hypertrophy in females treated at 100, 300 or 1000 mg/kg with respect to controls was observed.
- In thymus, a minimal decrease in cortical cellularity was seen in a male treated at 1000 mg/kg. The incidence and severity of decreased cortical cellularity were higher in females given 300 or 1000 mg/kg when respect to controls.
- Minimally to slightly decreased marginal zone cellularity in spleen was observed in one male and one female given the test item at 1000 mg/kg.

All other histological findings were considered 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.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

mortality

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

In conclusion, the effects of oral (gavage) administration of Lumière Blue PTM 0154N 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:
- Mortality, lower body weighs and histopathological findings in kidneys (tubular basophilia) were recorded at 300 and 1000 mg/kg/day with respect to Control. Based on these findings and on the fact that litter loss does not seem to be dose-related, the No Observed Adverse Effect Level (NOAEL) could be established at 100 mg/kg/day, as the increased incidence of adrenal cortical hypertrophy with respect to controls is considered mainly a response to stress.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study conducted in 2017

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

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008 

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.  

Based on available data on repeated dose toxicity, the substance is not classified for specific target organ toxicity following repeated exposure according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.

Substance is not classified as effects seen at 300 mg/kg/day were considered minimal.

The microscopic observation reveals minimal diffuse tubular basophilia in the kidneys of females given 300 or 1000 mg/kg/day, consistent with tubular degeneration and regeneration, and thus considered adverse.