1-[bis[3-(dimethylamino)propyl]amino]propan-2-ol

Endpoint summary

Description of key information

Repeated dose toxicity - oral:
A GLP compliant 90-day repeated dose toxicity study according to OECD guideline 408 was performed in rats via oral gavage. Dose levels tested were 10, 25 and 75 mg/kg. The NOEL for females was considered to be 25 mg/kg bw/day and 10 mg/kg bw/day for males.

Repeated dose toxicity - dermal/inhalation: No reliable data were available for these exposure routes. Therefore, no NOAEL for these routes of administration was established.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-13 to 2016-01-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffcat ZR 50
- Substance type: clear colourless liquid
- Physical state: liquid
- Analytical purity: 97.9%
- Impurities (identity and concentrations): 0.04% water
- Purity test date: no data
- Lot/batch No.: 1203-2014
- Expiration date of the lot/batch: 2016-12-01
- Stability under test conditions: stable for at least twenty-one days
- Storage condition of test material: at room temperature, in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: sixty male and sixty female Wistar Han™:RccHan™:WIST strain rats, obtained from Harlan Laboratories Limited, Oxon, UK
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: males: 184 to 226 grams, females: 159 to 202 grams
- Fasting period before study: no data
- Housing: in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Certificates of analysis of the batches of diet were provided.
- Diet (e.g. ad libitum): ad libitum, pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo Research Limited, Oxon, UK) was used.
- Water (e.g. ad libitum): ad libitum, mains drinking water supplied from polycarbonare bottles attached to the cage.
- Acclimation period: nine days, during which time their health status was assessed. The animals were examined for sings of ill-health or injury at receipt.
- The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12, low intensity fluorescent lighting, controlled

IN-LIFE DATES: From: 2015-07-03 to: 2015-10-30

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was prepared at the appropriate concentrations as a solution in distilled water.
- The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least twenty-one days.
- Formulations were prepared weekly and stored at approximately 4 ºC in the dark.
- The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

VEHICLE
- Concentration in vehicle: 0, 1, 2.5 and 7.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test item formulation were taken and analyzed on five occasions for concentration of Jeffcat ZR 50 at Envigo Research Limited, Shardlow, UK, Analytical Services. The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

daily

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

actual ingested

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were chosen based on the results of previous toxicity work (provided by the Sponsor)
- Rationale for selecting satellite groups: no data
- Post-exposure recovery period in satellite groups: 28 days following termination of treatment

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment-free period, animals were observed daily.
- All animals were examined for overt signs of toxicity, ill-health or behavioral change.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of treatment and at weekly intervals thereafter for all non-recovery animals.
- The following parameters were observed: gait, tremors, twitches, convulsions, hyper/hypothermia, skin color, respiration, palpebral closure, bizarre/abnormal/stereotypic behavior, salivation, pilo-erection, exophtalmia, lachrymation, urination, defecation, transfer arousal, tail elevation.

BODY WEIGHT: Yes
- Time schedule for examinations: day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION: yes
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and before termination of treatment (during week 12)
- Dose groups that were examined: all non-recovery animals
- Examinations included observation of the anterior structures of the eye following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a ophthalmoscope was performed.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (day 90) for all non-recovery animals, at the end of the treatment-free period (day 118) for all recovery group animals.
- Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on days 91 and 119.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals
- Parameters examined: hemoglobin; erythrocyte count; hematocrit; erythrocyte indices: mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration; total leukocyte count; differential leukocyte count: neutrophils, lymphocytes, monocytes, eosinophils, basophils; platelet count; reticulocyte count (slides were prepared but not assessed), prothrombin time was assessed by ‘Innovin’ and activated partial thromboplastin time was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (day 90) for all non-recovery animals, at the end of the treatment-free period (day 118) for all recovery group animals.
- Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on days 91 and 119.
- Animals fasted: No
- How many animals: all animals
- The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: urea, glucose, total protein, albumin, albumin/globulin ratio (by calculation), sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin, bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of the treatment and at weekly intervals thereafter for all non-recovery animals, during week 12 on all non-recovery animals
- Dose groups that were examined: all non-recovery animals
- Functional performances, assessment of sensory reactivity to different stimuli during week 12
- Motor activity: twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
- Forelimb/Hindlimb grip strength: an automated grip strength meter was used. Each nonrecovery animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
- Sensor reactivity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests. The following parameters were observed: grasp response, vacuolization, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex, startle reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- On completion of the dosing period or in the case of recovery group animals, at the end of the treatment-free period all animals were killed by intravenous overdose of a suitable barbiturate followed by exsanguination.
- All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS:
- The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen , testes , thymus, uterus

HISTOPATHOLOGY: Yes
- Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint) (retained only and not processed), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, colon, duodenum, epididymides (preserved in modified Davidson's fluid), esophagus, eyes (fixed in Davidson's fluid), gross lesions, heart, ilieum (including Peyer's patches), jejunum, kidneys, liver, lungs (with bronchi) (inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (mandibular and mesenteric), muscle (skeletal), ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin, spinal cord (cervical, mid thoracic and lumbar), spleen, stomach, testes (preserved in modified Davidson's fluid), thymus, thyroid/parathyroid, tongue (retained only and not processed), trachea , urinary bladder, mammary gland, uterus (with cervix), vagina.
- All tissues from non-recovery control and 75 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In
addition, sections of testes from all non-recovery control and non-recovery 75 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
- Since there were indications of treatment-related changes in the liver and spleen, examination was subsequently extended to include similarly prepared sections of the liver and spleen (both sexes) from animals in the low, intermediate and recovery dose groups. In addition, the liver was sectioned at 10μm for one control male, one control female, two non-recovery high dose males and females and two recovery high dose males and females and was stained with Oil Red O (ORO).

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: grip strength, motor activity, body weight change, hematology, blood chemistry, absolute organ weights, body weight-relative organ weights.
Data were analyzed using the decision tree from the Provantis TM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs detected that were considered to be related to test item toxicity. There were isolated incidences of three control males showing fur loss. As the control animals did not recieve any test item, the observation of fur loss was of no toxicological importance. One male treated with 75 mg/kg bw/day showed signs of chromodacryorrhea which later resulted in staining around the eyes. This observation was considered to be an isolated incident and to be of no toxicological importance.
See data tables for detailed information.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on study

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- There was an overall reduction in body weight gain in animals of either sex treated with 75 mg/kg bw/day and during weeks 7, 8 and 11, males from this treatment group showed a statistically significant reduction in group mean body weight gains. From week 7, weight gains in these males were generally lower than controls for the remainder of the study. A statistically significant reduction in weight gain during weeks 4 and 8 were evident in females from this treatment group and during week 11, these females showed actual body weight losses.
- During the recovery phase, body weight gains for either sex were comparable to recovery controls.
- No toxicologically significant effects were detected in animals of either sex treated with 10 or 25 mg/kg bw/day.
- Males treated with 25 mg/kg bw/day showed a statistically significant reduction in body weight gain during week 11 whilst females from this treatment group showed a statistically significant reduction in body weight gain during week 8. Body weight gains for the remainder of the study for these animals were comparable to controls therefore in isolation the intergroup differences were considered not to be of toxicological significance.
See data tables for detailed information.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food conversion efficiency was lower for animals of either sex treated with 75 mg/kg bw/day during Weeks 8 and 11 for males, and Weeks 4, 8 and 11 for females; these reductions correlated with the reduced body weight gains.
See data tables for detailed information.

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following intergroup differences were considered not to be of toxicological significance. Non recovery males from all treatment groups showed a statistically significant reduction in neutrophils. Non recovery females from all treatment groups showed a statistically significant increase in erythrocyte count. A true dose related response was not evident in either sex and the majority of individual values were within background control ranges for both parameters.
There was a statistically significant reduction in mean corpuscular hemoglobin evident in females treated with 75 and 25 mg/kg bw/day. Females treated with 75 mg/kg bw/day also showed a statistically significant reduction in mean corpuscular hemoglobin concentration. The majority of individual values were within background control ranges for both parameters.
Recovery females treated with 75 mg/kg bw/day showed statistically significant increases in erythrocyte count and eosinophils. These females also showed statistically significant
reductions in mean corpuscular hemoglobin and mean corpuscular volume. The majority of individual values were within the background control ranges.
See data tables for detailed information.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Both non recovery males and females treated with 75 mg/kg bw/day showed a statistically significant increase in alanine aminotransferase and aspartate aminotransferase when compared with controls. In recovery animals at this dose level, a similar effect was evident in these parameters. The majority of individual values were above the background control ranges and correlated with the histopathological findings detected in the liver.
All treated non recovery males showed a statistically significant reduction in total protein. Although the majority of individual values were within background control ranges this reduction can be correlated with the histopathological findings detected in the liver. No such effects were detected in females treated with 25 or 10 mg/kg bw/day.
Males treated with 10 mg/kg bw/day showed a statistically significant increase in glucose levels. The majority of individual values were above the background control ranges. A true dose relationship was not evident and there was no effect on the other treatment groups. Therefore this was considered to be of no toxicological importance. Recovery males treated with 75 mg/kg bw/day showed a statistically significant reduction in phosphorus. In the absence of a similar effect detected in non-recovery males at the end of the treatment period, the intergroup difference was considered of no toxicological significance.
See data tables for detailed information.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no effects observed on thyroid after histopathological examination.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no toxicological significant effects detected in the organ weights measured.
- Non-recovery males from all treatment groups showed a statistically significant increase in group mean thymus weights both absolute and relative to terminal body weight. There was no dose relationship present and the majority of individual values were within the background control ranges. There were no histopathological findings detected in the thymus therefore this was considered to be of no toxicological importance.
- At 75 and 25 mg/kg bw/day, non-recovery males showed a statistically significant increase in kidney weight both absolute and relative to terminal body weight. The majority of individual values were within the background control ranges and a true dose related response was not evident. There were no histopathological findings detected in the kidney therefore this was considered to be of no toxicological importance.
- There were statistically significant increases in both testes and epididymides weights from the non-recovery males treated with 75 mg/kg bw/day. The majority of individual values for each tissue were within the background control ranges and there were no histopathological findings detected. Therefore the intergroup differences were considered to be of no toxicological importance.
- Females treated with 10 mg/kg bw/day showed a statistically significant reduction in liver weight both absolute and relative to terminal body weight. In the absence of a similar effect in males or in females at 25 or 75 mg/kg bw/day, the intergroup difference was considered not to be of toxicological significance.
- Recovery females treated with 75 mg/kg bw/day showed a statistically significant increase in absolute and relative adrenal weight. In the absence of a similar effect detected in nonrecovery females at the end of the treatment period or any associated histopathological correlates, the intergroup difference was considered not to be of toxicological significance.
- Recovery females treated with 75 mg/kg bw/day also showed a statistically significant increase in liver weight both absolute and relative to terminal body weight. Although histopathological changes were evident in these females, the majority of the individual organ weights were within historical control ranges. In the absence of a similar effect in non-recovery females at the end of the treatment period, the intergroup difference was considered to be of limited toxicological significance.
See data tables for detailed information.

Gross pathological findings:

no effects observed

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All treated male groups showed a statistically significant increase in grip strength values during the final forelimb Test. There was no dose relationship or any associated clinical signs to suggest neurotoxicity. This only occurred in one out of the three tests therefore the intergroup differences were considered to be of no toxicological importance.
See data tables for detailed information.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Two males and four females treated with 75 mg/kg bw/day, two females treated with 10 mg/kg bw/day and four control females had reddened lungs at necropsy. There were no histopathological findings detected in this tissue and in view of the fact that the finding was also presented in controls, the macroscopic abnormalities were considered to be of no toxicological importance.

LIVER: There was centrilobular hydropic degeneration (presenting as enlarged vacuolated cytoplasm with centrally located nuclei; Oil-Red-O negative for fat) present from minimal to moderate in all non-recovery animals treated with 75 mg/kg bw/day. A mixed inflammatory infiltrate was present in the centrilobular area, minimal or mild in 8/10 males and all females treated with 75 mg/kg bw/day and at a minimal level in 2/10 males treated with 25 mg/kg bw/day. Single cell necrosis (minimal, centrilobular) was present in 7/10 males and 5/10 females treated with 75 mg/kg bw/day and in 2/10 males treated with 25 mg/kg bw/day. After the recovery period centrilobular hydropic degeneration was still apparent in 4/10 males and 2/10 females. Mixed inflammatory infiltrate in the centrilobular area was present in 5/10 males and females. Centrilobular single-cell necrosis at a minimal level was still present in 4/10 males and 5/10 females.

SPLEEN: Vacuolated macrophages were noted at a minimal or mild level in the spleen of 7/10 males and in all females treated with 75 mg/kg bw/day and at a minimal level in one male treated with 25 mg/kg bw/day. After the recovery period, vacuolated macrophages persisted in the spleen of one male and 5/10 females.
See data tables for detailed information.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects were detected in females treated with 25 mg/kg bw/day.

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No effects were detected in males treated with 10 mg/kg bw/day.

Key result

Critical effects observed:

no

Analytical verification of test item formulation:

The formulations investigated during the study were found to comprise test item in the range of 91 to 102% and, thus, the required content limit of +/- 10% with reference to the nominal content was met.

In addition, the test item was found to be stable in the formulations when kept for 12 and 21 days in the refrigerator (4°C) due to results which met the variation limit of 10% from the time-zero mean.

In conclusion, the results indicate that the accurate use of the test item and distilled water as vehicle during this study. The formulations were found to be homogeneously prepared (visual inspection) and sufficient formulation stability under storage conditions was proven.

Conclusions:

The oral administration of the test substance to rats by gavage, at dose levels of 10, 25 and 75 mg/kg bw/day resulted in reduced body weight gain at 75 mg/kg bw/day and treatment-related microscopic changes in the liver and spleen in either sex treated with 75 mg/kg bw/day and in males treated with 25 mg/kg bw/day. No such effects were detected in females treated with 25 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day, therefore the ‘No Observed Effect Level’ (NOEL) for females was considered to be 25 mg/kg bw/day and 10 mg/kg bw/day for males. Based on the proposed mechanism of action of osmotic effects and the observation of transient vacuolization in other aliphatic amines, this observation of vacuolization is considered also to be transient and non-adverse and as such the effects are of minimal toxicological importance. Regarding necrosis observed in the liver, the observation was described as “Centrilobular single-cell necrosis at a minimal level”. This description does not meet the criteria of “multi-focal or diffuse” as stated in the ECHA Guidance on the Application of the CLP Criteria for consideration of STOT RE classification. Therefore, these effects do not, by themselves or together, indicate “significant” toxicity in relation to Specific Target Organ Toxicity Repeated Exposure (STOT RE) classification in GHS. Since these observations in the OECD 408 study are considered not to support STOT RE classification, there is no need for this classification.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-06-07 to 2012-08-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well documented GLP study performed according to OECD Guideline 422. However, dose formulations were not analyzed.

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:

yes

Remarks:

No dose formulation analysis has been performed

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffcat ZR-50
- Substance type: Clear orange liquid
- Physical state: Liquid
- Lot/batch No.: PFW100119
- Storage condition of test material: Room temperature, 20.4 to 22°C
- Composition & purity: documented by the Sponsor, communicated to Calvert as Certificate of Analysis
- Stability: no information is provided

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan
- Age at study initiation: A minimum of 13 weeks old at initiation of cohabitation
- Weight at study initiation: 345-395 grams for the males and 211-258 grams for the females at initation of cohabitation
- Fasting period before study: No
- Housing: Upon arrival and until randomization, males and females were group-housed, sexes separate. Following randomization and until cohabitation, males and females were housed individually. During cohabitation, one female was placed with a male breeder from the same group. Following cohabitation, males and females were housed individually. No later than gestation day 17, mated female animals were placed in totes with bedding. The room in which the animals were kept were documented in the study records. No other species were kept in the same room.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Study animals were acclimated to their housing for a minimum of 7 days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 to 21.4 °C
- Humidity (%): 40.1 - 71.5%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights were turned on during the dark cycle to accommodate blood sampling or other study procedures.

Route of administration:

oral: gavage

Vehicle:

other: deionized water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Dose preparation: The test article formulations were prepared weekly or additionally as needed by diluting the test article in vehicle (w/v) to reach the proper concentrations.
- Dose formulation samples: On the first day of dosing, at the beginning of cohabitation and at the last day of dosing, duplicate 1 mL samples were obtained from top, middle, and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test article in vehicle. These samples were stored at room temperature, approximately 20.4 to 22°C. Dose formulation samples were not analysed and discarded at the finalisation of the study.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Male animals were dosed for a total of 35 days (starting two weeks prior to the cohabitation period). Treatment continued for the males during the same-group cohabitation period and until the day before scheduled euthanasia on day 21 of cohabitation. Female animals were dosed once daily for 15 days prior to cohabitation, during cohabitation, throughout pregnancy and up to including day 3 of lactation.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

actual ingested

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

actual ingested

No. of animals per sex per dose:

10 for all dose groups and 5 for the recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based upon previously conducted toxicity studies.
- Oral route was chosen as it is the route affording the maximum exposure, based on results of previously conducted acute studies

Positive control:

Not applicable

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Throughout the treatment phase, a minimum of twice daily, prior to dose administration and a minimum of once following dosing. On non-dosing days, a minimum of once daily.

BODY WEIGHT: Yes
- Males: Animals were weighed at the time of randomization/selection, on the first day of dosing and weekly thereafter. A fasted terminal body weight was recorded prior to scheduled euthanasia.
- Females: Animals were weighed at the time of randomization/selection, on the first day of dosing, weekly thereafter, and on gestation days 0, 4, 7, 14 and 20, 23 and 26, and on day 0 and 4 of lactation. A fasted terminal body weight was recorded prior to scheduled euthanasia.

FOOD CONSUMPTION:
- Males and females: Full feeder weights and/or feeder weigh backs were recorded once weekly prior to cohabitation, on gestation days 0-4, 4-7, 7-10, 10-14, 14-17, 17-20, 20-23 and 23-26 and on day 0 and 3 lactation. During cohabitation food consumption was not recorded.

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

CLINICAL PATHOLOGY EVALUATION (groups 1-6)
- Sample collection: Blood samples for evaluation of serum chemistry, hematology and coagulation parameters were collected from five animals/sex in all groups prior to terminal sacrifice. Animals were anesthetized by CO2 inhalation prior to blood collection. Immediately following exsanguination by cardiocentesis for terminal blood collection, rats were returned to the CO2 chamber to ensure euthanasia. Animals were fasted overnight (approximately 12-24 hours) prior to blood collection for clinical pathology evaluation.

HAEMATOLOGY: Yes
- Method of collection: cardiocentesis
- Anticoagulant: K2-EDTA
- Parameters analyzed: red blood cell count and morphology, white blood cell count*, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, hematocrit, hemoglobin, reticulocyte count
*: total and different white blood cell counts, including neutrophils, basophils, eosinophils, monocytes, lymphocytes and large unstained cells
- Coagulation:
Method of collection: cardiocentesis
Anticoagulant: sodium citrate
Coagulation parameters: activated partial thromboplastin time, prothrombin time

CLINICAL CHEMISTRY: Yes
Method of collection: cardiocentesis
Anticoagulant: none
Parameters analyzed: Alanine aminotransferase, albumin, albumin/globulin ratio (calculated), alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatinine, globulin (calculated), glucose, phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea nitrogen

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
The functional observational battery was assessed for five rats/sex/group once during the study (toward the end of the dosing periods). Each rat was placed in a fixed environment considering of a Plexiglas enclosure, fitted with a lid. The enclosure was placed on absorbent paper which detects excretions. In this environment, rats were free to move about. The rats were observed for signs of pharmacological or toxicological activity following treatment and the results recorded.
Observations for the following symptoms were made: abnormal posture, ataxia, awareness reaction, body tremors, corneal reflex, decreased abdominal tone, decreased grip strength, decreased respiration, excretion, immobility, increased secretion, irritability, loss of righting, motor activity, nociceptive (pain) response, piloerection, pinnal reflex, pupil size, seizures/convulsions, startle response, sterotypy, vocalization

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Tissue collection and preservation (groups 1-6):
All tissues for all adults were examined. For all animals necropsied, the tissues listed below were preserved in 10% neutral buffered formalin (except for the epididymides and testes that were retained in modified Davidson's fixative for optimum fixation).
Tissues collected: Cardiovascular: aorta*, heart; digestive: salivary gland(s), tongue, esophagus, stomach*, small intestine* (duodenum, jejunum, ileum); urogenital: kidneys*, urinary bladder*, ovaries*, uterus*, cervix*, vagina*, testes*, epididymides*, prostate*, seminal vesicles*; endocrine: adrenals*, pituitary, thyroid/parathyroid*; large intestine* (cecum, colon, rectum), pancreas, liver*; respiratory: trachea*, larynx, lung with mainstem bronchus*; lymphoid/hematopoietic: sternum with bone marrow*, thymus*, spleen*, lymph nodes* (mandibular, mesenteric); skin/musculoskeletal: skin, mammary gland, skeletal muscle, femur with articular surface; nervous/special sense: eye with optic nerve, sciatic nerve*, brain*, spinal cord - cervical*, spinal cord - midthoracic*, spinal cord - lumbar*, lacrimal glands; other: unique animal identifier (not for evaluation), gross findings*

ORGAN WEIGHTS:
- For all male group 1-6 animals, the following organs were weighed before fixation, after dissection of excess fat and other excess tissues. Organ weights were not recorded for animals found dead.
Organs weighed: epididymides, testes,
- For five male and female groups 1-6 animals, at scheduled sacrifice, the following organs (when present) were weighed before fixation, after dissection of excess fat and other excess tissues. Organ weights were not recorded for animals found dead. Paired organs were weighed together unless gross abnormalities were present, in which case they were weighed separately.
Organs weighed: adrenals, heart, kidneys, liver, thymus, spleen, brain
- Organ to body weight ratios were calculated (using the final body weight obtained prior to necropsy), as well as organ to brain weight ratios.


HISTOPATHOLOGY: Yes
- Histology (groups 1-6):
Tissues for evaluation were processed to paraffin blocks and prepared to slides. Slides were stained with hematoxylin and eosin. Occasionally, other stains were required by the study pathologist to aid in the diagnosis of lesions; these were documented in the final report.

Slides were prepared for five animals/sex in group 1, 4, 5 and 6 for all tissues marked with * above.

If test article-related lesions were noted, additional slides were prepared on those tissues from groups 2 and 3 at additional cost to the Sponsor, following the Sponsor's consent.

Statistics:

Statistical evaluation was performed on in-life, clinical pathology, and organ weight numerical data. For in-life and clinical pathology parameters, the software determined statistical significance by the following decision tree. First, the homogeneity of the data was determined by Barlett's test. If the data were homogeneous, a one-way analysis of variance was performed to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test were used to determine the degree of significance from the control means (p<0.05, p<0.01 and p<0.001). If the data is non-homogeneous, the Kruskal-Wallis non-parametric analysis was performed to assess statistical signficance. If statistically significant differences between the means were found (p<0.05, p<0.01 and p<0.001), the Mann-Whitney U-Test was used to determine the degree of significance from the control means (p<0.05, p<<0.01 and p<0.001). For necropsy organ weight data, the evaluation of the equality of means were made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, Dunnett's test was used to determine the degree of significance from the control means (p<0.05 and p<0.01).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Females: Clinical observations in Group 6 animals were limited to two animals with instances of food crumbling between days 2-12 of the dosing phase. In addition, one group 6 animal had piloerection between days 12-13 and staining on the head (cranial) between days 12-15.

Males: Male Group 6 animal #0346 had noisy respiration between days 3-4. On day 28, it also exhibited piloerection, had red discharge around the muzzle and nares, and had stained forepaws. All other male Groups 5-6 animals appeared normal during the dosing and recovery phase.

Adult clinical observations:
Females: All female group 1 animals appeared normal during the study. Female group 2 findings were limited to animal #0363 with hair loss around the muzzle with associated moderate, occasional swelling between cohabitation phase day 1 and gestation phase day 13. Three group 3 animals exhibited instances of food crumbling on some days between dosing day 2 and gestation day 5. All but one group 4 animals exhibited clinical signs including but not limited to, pale skin, food crumbling, loose feces, noisy respiration, nasal discharge and/or piloerection. Some of these clinical signs were seen as early as dosing phase day 2 in some animals and lasted up to lactation phase day 3.
Males: All male groups 1-4 animals appeared normal during the dosing and cohabitation phase of the study. Only the incidental missing of the upper incisors was observed in one group 2 and two group 3 animals.

Mortality:

no mortality observed

Description (incidence):

All male and female animals survived until their scheduled sacrifice on day 57 (17 days after their respective last day of dosing).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Females: Statistically significantly lower body weights were observed between days 42-56 in unmated Group 6 females compared to concurrent control Group 5 animals. In addition, body weight gains were statistically significantly higher for Group 6 on Dosing Day 42.
Males: Statistically significantly lower body weights were observed between days 35-56 in Group 6 males compared to Group 5. In addition, body weight gains were statistically significantly lower for Group 6 on dosing day 21.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Females: Female Group 6 food consumption was statistically significantly increased during the recovery phase on days 49 and 56.
Males: Food consumption was statistically significantly reduced among Group 6 animals on days 7, 35 and 42, and statistically significantly increased at the end of the recovery phase on day 56.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Females: Similar to mated Group 4 animals, at the end of the recovery period on day 57 unmated female Group 6 leukocyte counts (WBC) were statistically significantly increased and red blood cell (RBC) hemoglobin (HGB) and hematocrit values (HCT) were statistically significantly decreased. In addition, Group 6 absolute basophil (#BASO) and large unstained cell levels (#LUC) were statistically significantly increased. All red blood cells in both dose groups were normocytic and normochromic.
Males: Male Group 6 platelet values (PLT) were statistically significantly increased. All red blood cells in both dose groups were normocytic and normochromic.
Coagulation:
Females: Group 6 had statistically significantly decreased prothrombin times (PT) when compared to Group 5 animals. However, the slightly reduced PT were not considered adverse.
Males: No statistically significant effects on prothrombin times (PT) and activated partial thromboplastin times (APTT) were detected at the end of the recovery phase on day 57.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Females: Group 6 aspartate aminotransferase values (AST) were 33% increased. In addition, Group 6 inorganic phosphorus (PHOS), cholesterol (CHOL) and chloride levels (CL) were statistically significantly increased, and creatinine levels (CREAT) and albumin globulin ratios (A/G) were statistically significantly decreased.
Males: Significant changes in liver enzyme chemistry were also seen in Group 6 animals at the end of the recovery phase on day 57. Group 6 aspartate aminotransferase values (AST) were 107% and alanine aminotransferase values (ALT) were 156% increased, respectively. In addition, Group 6 total protein (TP), globulin (GLOB) and albumin level (ALB) were all statistically significantly decreased.

Since vacuolation and fibrosis was still present in the liver of all male and female Group 6 animals, the associated changes in clinical chemistry parameters were still present at the end of the recovery period on day 57.

Endocrine findings:

no effects observed

Description (incidence and severity):

No histopathological effect was observed in adrenals, pituitary and parathyroid/thyroid glands.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Females: There were no treatment-related findings for any of the qualitative functional observational battery tests peformed on dosing day 37.
Males: There were no treatment-related findings for any of the qualitative functional observational battery tests performed on dosing day 37.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females: Similar to the groups 3-4 findings above, unmated female group 6 absolute liver weights and liver-to-body and liver-to-brain weights were statistically significantly increased. Female group 6 liver weights were 116% above control group 5 values. In addition, group 6 kidney weights were statistically significantly increased.
Males: Male group 6 absolute liver weights, liver-to-body and liver-to-brain weights were statistically significantly increased. Group 6 liver weights were 59% above control group values. In addition, group 6 body weights were statistically significantly decreased, and group 6 testes-, spleen- and kidney-to-body weight ratios were statistically significantly increased.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Females: At the end of the 16 day recovery phase all female group 6 animals had enlarged and pale livers at necropsy. Microscopically, these findings correlated to slight to moderate vacuolation of the hepatocytes, predominantly centrilobular.
Males: All male group 6 animals had enlarged and pale livers at necropsy. Microscopically, these findings also correlated to slight to moderate vacuolation of the hepatocytes, predominantly centrilobular. One male group 5 animal had an incidentally enlarged prostate with no microscopic correlation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test article toxic effects were still present in the parathyroid glands, trachea, lungs, liver and spleen of males and females euthanized 15 days after the last day of dosing with the test substance at 250 mg/kg.
Females and males: Parathyroid gland: minimal to slight vacuolation of the parathyroid chief cells was still present in 4 of 5 males and 5 of 5 females from the highest dose group (group 6).
Trachea: minimal vacuolation of the tracheal epithelial cells still occurred in 1 of 5 males and 2 of 5 females from the highest dose group (group 6).
Lungs: minimal to slight epithelial vacuolation was still present within the bronchi and bronchioles in all males and females previously treated with the test substance at 250 mg/kg (group 6). In addition, minimal to slight vacuolation of the media layer was still present in the pulmonary blood vessels in 2 of 5 males and 5 of 5 females from the previously treated highest dose group (group 6).
Liver: vacuolation of the hepatocytes and parenchymal fibrosis were still present in the liver of all male and female rats previously treated with the test substance at 250 mg/kg (group 6). The hepatocyte vacuolation was still predominantly centrilobular but with reduced severity grade when compared to livers from mated animals. The severity grade ranged from slight to moderate. Fibrosis was still present within the centrilobular regions and was increased in severity when compared to livers from mated animals. In addition, to increased severity in group 6 animals, fibrosis often bridged among centrilobular areas, a feature not seen in group 4 animals.
Spleen: foam cells were still present in the splenic red pulp of 1 of 5 males and 5 of 5 females previously treated with the highest dose of the test substance (group 6). However, the severity was decreased. Minimal to slight decrease of the cellularity of the marginal zone was still present in 3 of 5 male and 1 of 5 female rats previously treated with the test substance at 250 mg/kg (group 6).

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Male and female rats were dosed orally once daily at 0, 25, 100 and 250 mg/kg to test for potential toxic effects/disturbances resulting from the administration of the test substance. Male animals were dosed for a total of 35 days (starting two weeks prior to the cohabitation period). Treatment continued for the males during the same-group cohabitation period and until the day before scheduled euthanasia on Day 21 of cohabitation. Female animals were dosed once daily for 15 days prior to cohabitation, during cohabitation, throughout pregnancy and up to including Day 3 of lactation. Male and female animals dosed at 100 and 250 mg/kg showed significant effects of toxicity. One female Group 4 dam dosed at 250 mg/kg was found dead on Lactation Day 1 and all its neonates were found dead. All other female animals survived until their scheduled sacrifice. Besides adverse clinical signs and effects on body weight and food consumption, Groups 3 and 4 animals exhibited adverse microscopic and macroscopic liver findings, effects on liver weights, BUN levels, and liver enzymes including AST and ALT. Microscopic liver findings included enlargement and pallor secondary to hepatocyte vacuolation and centrilobular fibrosis, which severity increased proportionally with dose levels. In addition at 100 and 250 mg/kg test substance induced cytoplasmic vacuolation also occurred in the chief cells of the parathyroid gland (high dose groups only), tracheal, bronchial and bronchiolar epithelium, and within the media of the pulmonary vasculature. Additional findings included foam cells in the splenic red pulp (high dose groups only), decreased cellularity of the splenic marginal zone (high dose groups only), and erosion on the glandular gastric mucosa. At 25 mg/kg microscopic findings were limited to the gastric mucosa of one animal and the liver of another animal.
Additional five animals/sex/groups, dosed orally once daily at 0 and 250 mg/kg, were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity. After the last day of dosing on Day 40 the satellite animals stayed on study for an additional 16 days without dosing to observe the reversibility, persistence or delayed occurrence of systemic toxic effects. All male and female animals survived until their scheduled sacrifice on Day 57 (17 days after their respective last day of dosing). Clinical observations in Group 6 animals included instances of food crumbling, piloerection, staining on the head (cranial) and red discharge around the muzzle and nares with associated stained forepaws. Body weights and food consumption was variable between male and female satellite animals. Similar to Groups 3 and 4 animals, unmated Group 6 satellite animals dosed at 250 mg/kg exhibited adverse microscopic and macroscopic liver findings, effects on liver weights and liver enzymes. All microscopic findings described for mated Groups 3 and 4 animals above were also present after the 16-day recovery period in the unmated Group 6 animals. The severity was similar or reduced grade except for hepatic fibrosis, which was more preeminent in livers from recovery groups.

Key result

Dose descriptor:

NOAEL

Effect level:

< 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Maternal and paternal hepatotoxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw (total dose)

System:

hepatobiliary

Organ:

liver

Treatment related:

not specified

Conclusions:

Based on the results of this study, the no observed adverse effect level (NOAEL) for male and female rats exposed to the test substance is considered to be less than 25 mg/kg/day, based on maternal and paternal hepatotoxicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP-compliant study, according to OECD guideline

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity - oral:

90day repeated dose toxicity study :

A 90day repeated dose toxicity study is performed in rats. Male and female animals were dosed via oral gavage at dose levels of 0, 10, 25 or 75 mg/kg/day, according to OECD guideline 408 (Edwards, 2016; K1, GLP).

The NOEL (NOAEL) for oral repeated dose toxicity is determined at 10 mg/kg/day (males) and 25 mg/kg/day (females). No toxicological relevant effect was seen in mortality, clinical signs, water consumption, ophthalmology, hematology, functional performance, sensory activity.

There was an overall reduction in body weight gain in males and females at 75 mg/kg/d (accompanied by lower food conversion efficiency), and in males dosed at 25 mg/kg/d. Males and females at 75 mg/kg/d showed a significant increase in clinical chemistry parameters like aspartate aminotransferase and alanine aminotransferase. Males treated at 75 mg/kg/d showed a statistically significant reduction in total protein. Females treated at 75 mg/kg/d showed a significant increase in liver weight. Toxicologically relevant findings in liver and kidney were observed. The primary observations in the study were 1) vacuolation in liver and spleen of males and females treated at 75 mg/kg bw/day and 2) centrilobular single-cell necrosis (Grade: minimal) in the liver of males and females at 75 mg/kg/day and males at 25 mg/kg/day. Regarding vacuolation in the liver and spleen, the vacuoles observed are considered transient and non-adverse based on scientific peer-reviewed literature regarding weakly basic aliphatic amines and supporting evidence from similar compounds within the same chemical family of aliphatic amines. Regarding necrosis observed in the liver, the observation was described as “Centrilobular single-cell necrosis at a minimal level”. This description does not meet the criteria of “multi-focal or diffuse”as stated in the ECHA Guidance on the Application of the CLP Criteria for consideration of STOT RE classification. Therefore, these effects do not, by themselves or together, indicate “significant” toxicity in relation to Specific Target Organ Toxicity - Repeated Exposure (STOT RE) classification in GHS. Since these observations in the OECD 408 study are considered not to support STOT RE classification, the substance is not classified.

Combined repeated dose toxicity with screening reproduction/developmental study

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test is performed in rats, in which male and female rats are exposed to 0 (vehicle), 25, 100, 250 mg/kg bw/d via gavage (OECD 422).

The NOAEL for oral repeated dose toxicity in Sprague-Dawley rats is considered to be < 25 mg/kg bw/day (actual dose received) for both sexes. Male and female animals dosed at 100 and 250 mg/kg showed significant effects of toxicity. One female group 4 dam dosed at 250 mg/kg was found dead on lactation day 1 and all its neonates were found dead. All other female animals survived until their scheduled sacrifice. Besides adverse clinical signs and effect on body weight and food consumption, groups 3 and 4 animals exhibited adverse microscopic and macroscopic liver findings, effects on liver weights, urea levels, and liver enzymes including AST and ALT. Microscopic liver findings included enlargement and pallor secondary to hepatocyte vacuolation and centrilobular fibrosis, which severity increased proportionally with dose levels. In addition at 100 and 250 mg test substance/kg induced cytoplasmic vacuolation also occurred in the chief cells of the parathyroid gland (high dose groups only), tracheal, bronchial and bronchiolar epithelium, and within the media of the pulmonary vasculature. Additional findings included foam cells in the splenic red pulp (high dose groups only), decreased cellularity of the splenic marginal zone (high dose group only), and erosion on the glandular gastric mucosa. At 25 mg/kg microscopic findings were limited to the gastric mucosa of one animal and the liver of another animal.

After the last day of dosing on day 40 the satellite animals stayed on study for an additional 16 days without dosing to observe the reversibility, persistence or delayed occurrence of systemic toxic effects.

All male and female animals survived until their scheduled sacrifice on day 57 (17 days after their respective last day of dosing). Clinical observations in group 6 animals included instance of food crumbling, piloerection, staining on the head (cranial) and red discharge around the muzzle and nares with associated stained forepaws. Body weights and food consumption was variable between male and female satellite animals. Similar group 3 and 4 animals, unmated group 6 satellite animals dosed at 250 mg/kg exhibited adverse microscopic and macroscopic liver findings, effects on liver weights and liver enzymes.

All microscopic findings described for mated groups 3 and 4 animals above were also present after the 16 day recovery period in the unmated group 6 animals. The severity was similar or reduced grade except for hepatic fibrosis, which was more preeminent in livers from recovery groups. In conclusion, the toxic effects seen at 250 mg/kg in mated group 4 animals were not reversible after a 16 day recovery period without dosing in the unmated group 6 satellite animals.

Repeated dose toxicity - dermal/inhalation:

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal or inhalation route of exposure.

Justification for classification or non-classification

Based on the proposed mechanism of action of osmotic effects and the observation of transient vacuolization in other aliphatic amines, this observation of vacuolization is considered also to be transient and non-adverse and as such the effects are of minimal toxicological importance. Regarding necrosis observed in the liver, the observation was described as “Centrilobular single-cell necrosis at a minimal level”. This description does not meet the criteria of “multi-focal or diffuse”as stated in the ECHA Guidance on the Application of the CLP Criteria for consideration of STOT RE classification. Therefore, these effects do not, by themselves or together, indicate “significant” toxicity in relation to Specific Target Organ Toxicity Repeated Exposure (STOT RE) classification in GHS. Since these observations in the OECD 408 study are considered not to support STOT RE classification, the substance is not classified.