Registration Dossier

Administrative data

Description of key information

repeated dose toxicity - oral: A key K1 study in male and female Wistar rats in a combined repeated dose toxicity test with reproductive/developmental screening was conducted according to OECD guideline 422 (Edwards, 2018). The NOAEL is established to be 500 mg/kg body weight/day.

repeated dose toxicity - dermal or 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 inhalation or dermal route of exposure.

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:
2017-07-13 - to final report date
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)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: (EC) No 440/2008
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: Tris (2-hydroxyethyl) methylammonium hydroxide
- CAS number: 33667-48-0
- Physical state/appearance: brown viscous liquid
- Batch No.of test material: D26-3-3-1
- Expiration date: 01 May 2019
- Purity: 47.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected as it is a readily available rodent species, historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Males: approx. 12 weeks
Females: approx.14 weeks
- Weight at study initiation:
Males: 266g - 359g
Females: 198 - 240g
- Fasting period before study: Not specified
- Housing:
- Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK)
- During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group.
- Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: ad libitum. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.)
- Water: ad libitum. Main drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 2017-08-07 To: 2017-10-08
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
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.
- Formulations were prepared weekly for the first two weeks of treatment (as only twelve day stability had been confirmed) and then every two weeks once twenty-one day stability data had been determined and stored at approximately 4 ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 15, 50, 100 mg/mL
- Amount of vehicle (if gavage): 5mL/kg. 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.
- Batch No.of test material: not specified
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The stability and homogeneity of the test item formulations indicated that the formulations were stable for at least 21 days.
- Samples of test item formulations were taken on three occasions and analyzed for concentration and the results indicate that the prepared formulations were within ± 11% of the nominal concentration.
Duration of treatment / exposure:
Males : Approx. 6 weeks
Females : up to 8 weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control)
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low dose); 36 mg/kg bw/day of THEMAH
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 3 (Intermediate dose); 119 mg/kg bw/day of THEMAH
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 4 (High dose); 237 mg/kg bw/day of THEMAH
No. of animals per sex per dose:
48 males and 48 females in total; 12 males and 12 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen ibased on the results of previous toxicity work including a fourteen day dose range-finding toxicity study in the rat (Envigo Study Number FT63PY).
- Treatmetn volume: 5 mL/kg body wei
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: immediately before dosing, soon after dosing, and one hour after dosing during the working week (except for females during parturition where applicable)
- Parameters: signs of toxicity, ill-health and behavioral change

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4, 7 and 14 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
- During the pre-pairing period: weekly food consumption was recorded for each cage of adults.
- For males after the mating phase: weekly food consumption was recorded for each cage of adults.
- For females showing evidence of mating: food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20.
- For females with live litters: food consumption was recorded for the periods covering post partum Days 1-4, 4-7 and 7-14.

FOOD EFFICIENCY: Yes
- For males: was calculated retrospectively throughout the study period (with the exception of the mating phase).
- For females during the pre-pairing phase: was calculated throughout retrospectively the study period (with the exception of the mating phase).
- Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: water intake was observed daily by visual inspection of water bottles for any overt changes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination (Day 43 for males and Day 13 post partum for females)
- Anaesthetic used for blood collection: no. Blood samples were obtained from the lateral tail vein.
- Animals fasted: no
- How many animals: five males and five females selected from each test and control group.
- Parameters examined: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic), Methemoglobin (Meth), Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) 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: prior to termination (Day 43 for males and Day 13 post partum for females)
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group.
- Parameters examined: Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++).

OTHER:
FUNCTIONAL OBSERVATION
Prior to the start of treatment and at approximately weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. These observations were performed on mated females on Days 4, 11 and 18 post coitum and for littering females on Days 4 and 12 post partum. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIORAL ASSESSMENT:
- Detailed individual clinical observations were performed for each animal using a purpose built arena.
- The parametes observed were : Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

FUNCTIONAL PERFORMANCE
- Motor activity: urpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period.
- Forelimb/Hindlimb Grip Strength: An automated meter was used. Each 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. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made.

SENSOR REACTIVITY
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
- Parameters observed : Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach.

THYROID HORMONE ANALYSIS
- serum and plasma samples were taken from all adult males and females at termination.


Sacrifice and pathology:
SACRIFICE
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45.
Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post partum.

GROSS NECROPSY
- Post mortem procedures were performed on all adult animals and included any animal found dead or killed in extremis during the study.
- A full external and internal examination

ORGAN WEIGHT
-For five males and five females selected from each test and control group.
-The following organs were dissected free from fat and weighed before fixation: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (weihed partially fixed), Prostate, Seminal vesicles (with coagulation gland), spleen, Testes, Thymus, Thyroid (weighed partially fixed with parathyroid), Uterus (weighed with cervix and oviducts).
-For all remaining animals, the following organs were weighed: Epididymides, Prostate, Seminal Vesicles (with coagulation gland), Ovaries, Pituitary (weighed after partial fixation), Thyroid (weighed after partial fixation with parathyroid), Uterus (weighed with Cervix).

HISTOPATHOLOGY
- From five males and five females selected from each test and control group had the following organs preserved in buffered 10% formalin.
- The tissues were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination.
- Tissues / organs collected: Adrenals, Aorta (thoracic), Bone and bone marrow (femur including stifle joint), Bone and Bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Cowpers glands, Duodenum, Esophagus, Eyes (retained in Davidson's Fluid), Glans penis, Gross lesions, Heart, Ileum (including peyer's patches), Jejunum, Kidneys, LABC (levator ani-bulbocavernous) muscle, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (with coagulation gland), Skin, Spinal cord (cervical, mid-thoracic and lumbar), Spleen, Stomach, Testes (retained in Modified Davidson's Fluid), Thymus, Thyroid/Parathyroid, Trachea, Urinary bladder, Uterus and Cervix (with oviducts), Vagina.
Statistics:
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).
Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing.
Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test.
Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the KruskalWallis test which if significant was followed by the Mann-Whitney "U" test.
Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were 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 apparent that were considered to be related to systemic toxicity of the test item. Isolated occurrences of increased salivation were noted in animals of either sex treated with 500 mg/kg bw/day (four males and three females) from Day 1 until Day 33 (males) and from Day 19 until Day 50 (females). Occasional instances of noisy respiration (eleven males and eleven females) was also noted in these animals from Day 1 until Day 44 (males) and in female animals from Day 1 until Day 52.
One female animal treated with 500 mg/kg bw/day also exhibited hunched posture and piloerection on Day 41. As these observations were noted around the time of parturition, in isolation, and similar findings were not apparent in any other high dose animals these clinical signs were considered to be incidental and unrelated to treatment with the test item.
Isolated occurrences of noisy respiration were noted in animals of either sex treated with 250 mg/kg bw/day (six males and nine females) from Day 8 to Day 30 (males) and from Day 10 to Day 50 (females). No clinical signs were apparent in any animal of either sex treated with 75 mg/kg bw/day. Signs of noisy respiration were also noted in one male and one female from the control group, this was frequently noted in the male animal from Days 7 to 45 and was noted on one day only in the female animal.
Increased salivation is commonly observed in this type of study and is generally considered to be due to an irritant/unpalatable nature of the test item and/or formulation. As such this observation is considered not to be specifically related to systemic toxicity of the test item. Signs of noisy respiration may represent a potential difficulty in dosing these animals which may have caused a slight reflux of the test item or vehicle and cannot be considered to be related specifically to toxicity of the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal treated with 250 mg/kg bw/day was found dead on Day 51. This occurred during the blood sampling procedure and was therefore considered unrelated to treatment with the test item. There were no further unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were considered to be no adverse effects on body weight development in treated male animals during the study.
Male animals treated with 500 mg/kg bw/day exhibited reductions in body weight gain during the first three weeks of treatment but without achieving statistical significance. Recovery was evident thereafter such that body weight gains exceeded control throughout the remainder of the treatment period. An overall body weight gain for these animals was only 7.2% lower than control at the end of the treatment period and as such this apparent effect was considered not to be toxicologically significant.
Males animals treated with 250 mg/kg bw/day exhibited reductions in body weight gain during the first three weeks of treatment but without achieving statistical significance (these gains were slightly higher than those noted in male animals treated with 500 mg/kg bw/day). Recovery was evident thereafter, such that body weight gains were comparable to or exceeded control. However, an overall body weight gain for these animals was only 5% lower than control at the end of the treatment period and as such this apparent effect was considered not to be toxicologically significant.
Male animals treated with 75 mg/kg bw/day exhibited a statistically significant (p<0.05) increase in body weight gain during the fifth week of treatment. An increase in body weight gain is considered not to be an adverse effect of treatment and therefore is considered not to be toxicologically significant.
There were considered to be no effects on body weight development in treated females during maturation and no adverse effects during gestation or lactation.
Females treated with 500 mg/kg bw/day exhibited a slight reduction (without achieving statistical significance) in body weight gain during Week 1 of gestation which resulted in a statistically significant (p<0.05) lower body weight than control on Day 7. However, recovery was evident during the remainder of the gestation phase such that body weight gains and overall body weight gains were comparable to control.
During the lactation phase of the study females from all treatment groups exhibited reductions in body weight gains when compared to control animals from Day 1 to Day 4 (but without achieving statistical significance). However, the body weight gains for the control animals appeared to be higher than would generally be expected and as such may have artificially accentuated the appearance of reduced body weight gain in the treated animals. Comparable body weight gains were then noted across all treatment groups (from Day 4) when compared with control. For females treated with 500 mg/kg bw/day, actual body weights were statistically significantly lower (p<0.05) than control on Days 4 and 14. Even with body weight gains being comparable to control after Day 4 of lactation, overall body weight gains for all treatment groups were 11%, 3% and 13% lower than control at dose levels of 75, 250 and 500 mg/kg bw/day respectively. As a dose relationship was not apparent in these animals, these intergroup differences are considered to be due to normal biological variation and unrelated to treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was considered to be no effects in food consumption in any treated male animals. There was considered to be no effects in food consumption in female animals during maturation and no adverse effects in food consumption in females during gestation or lactation.
Females treated with 500 mg/kg bw/day exhibited a statistically significant (p<0.05) reduction in food consumption from Days 7 to 14 of gestation. As the reduction noted was minimal this was considered not to be toxicologically significant.
There was considered to be no effects in food consumption for females treated with 75 and 250 mg/kg bw/day during gestation.
All treatment groups exhibited reductions in food consumption throughout lactation when compared to control (but without achieving statistical significance). However, the food consumptions for the control animals appeared to be higher than would generally be expected and as such may have artificially accentuated the appearance of reduced food intake in the treated animals. These intergroup differences are therefore considered to be due to normal biological variation and unrelated to treatment with the test item.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
At 500 and 250 mg/kg bw/day, food conversion efficiency for males appeared inferior to control during the pre-pairing phase of the study. At 75 mg/kg bw/day, food conversion efficiency for males appeared unaffected by treatment.
Food conversion efficiency for females during the pre-pairing phase of the study appeared unaffected by treatment at 75, 250 or 500 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were considered to be no effect on water consumption for any treated animal.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined.
Statistically significant increases in reticulocytes (p<0.01) were noted in males treated with 500 mg/kg bw/day and in female animals (p<0.05) treated with 250 or 500 mg/kg bw/day, a dose relationship was apparent in the female animals. However, as all values lay within the historical control data range and there were no histopathological correlates these findings were considered to be of no toxicological significance.
Statistically significant increases (p<0.05) in mean corpuscular haemoglobin concentration were noted in females treated with 75 mg/kg bw/day, two values were found to be higher than the historical control data range. As similar findings were not noted in female animals treated with 250 or 500 mg/kg bw/day these findings are considered to be incidental and not toxicologically significant.
No such findings were apparent in male animals treated with 75 or 250 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were considered to be no toxicologically significant effects detected in the blood chemical parameters examined.
Males from all treatment groups exhibited statistically significant increases (p<0.05) in glucose and statistically significant decreases (p<0.05) in alanine aminotransferase, dose relationships were not apparent. All values generally lay within the historical control data ranges for glucose, with the exception of one value from animals treated with 75 mg/kg bw/day which was slightly higher. All values generally lay within the historical control data ranges for alanine aminotransferase, however, two values from control animals and one value from animals treated with 75 mg/kg bw/day were higher. In the absence of any histopathological correlates these findings are considered to be incidental and not toxicologically significant.
Male animals treated with 500 mg/kg bw/day exhibited statistically significant decreases (p<0.05) in phosphorous with a dose relationship being apparent. Three of these values were lower than the historical control data range. However, as there were no histopathological correlates these findings are considered to be incidental and not toxicologically significant.
Females treated with 75 mg/kg bw/day exhibited a statistically significant increase (p<0.05) in albumin/globulin ratio. All values were within the historical control data ranges and as there were no histopathological correlates or any similar effects noted in animals treated with 250 or 500 mg/kg bw/day these findings are considered to be incidental and not toxicologically significant.
No such findings were apparent in females treated with 250 or 500 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
-Behavioral assessments
Sporadic instances of noisy respiration were noted in one male and one female animal treated with 250 mg/kg bw/day and in four males and two female animals treated with 500 mg/kg bw/day and was noted a maximum of three times per animal during the treatment period. One control male animal also exhibited noisy respiration on four separate occasions. There were no treatment-related changes in the behavioral parameters in animals of either sex treated with 75 mg/kg bw/day.

-Functional Performance Tests
There were considered to be no treatment related changes in functional performance considered to be related to treatment at 75, 250 or 500 mg/kg bw/day.
Male animals treated with 75 or 250 mg/kg bw/day exhibited a statistically significantly higher (p<0.05) hind limb grip strength. As these increases were only noted in one out of the three runs completed (Run 1), there were no clinical signs observed to signify a neurotoxic effect of the test item and a dose relationship was not apparent, therefore, these two findings were considered to be incidental and of no toxicological relevance.

-Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 75, 250 or 500 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were considered to be no toxicologically significant effects detected in the organ weights measured.
Male animals treated with 500 mg/kg bw/day showed a statistically significant increase (p<0.01) in epididymis weights both absolute and relative to terminal body weight. Four absolute values were outside of the normal historical control data range with nine values for relative organ weights outside of these ranges also. In the absence of any histopathological correlates, these findings were considered to be incidental and unrelated to treatment with the test item.
Male animals treated with 250 or 500 mg/kg bw/day exhibited statistically significant increases (p<0.05) in prostate weights both absolute and relative to terminal body weight. Two absolute values were higher than the historical control data range for animals treated with 250 mg/kg bw/day, whereas all values were within the historical control data range for animals treated with 500 mg/kg bw/day. Three and two relative value for animals treated with 250 or 500 mg/kg bw/day respectively were higher than the normal historical control data range. In the absence of any histopathological correlates, these findings were considered to be incidental and unrelated to treatment with the test item.
Female animals treated with 250 mg/kg bw/day showed statistically significant decreases (p<0.05) in heart weights both absolute and relative to terminal body weight. Three values for control animals for both absolute and relative organ weights were higher than the historical control data range, whereas only one value for absolute and relative organ weights was outside of the historical control range for animals treated with 250 mg/kg bw/day. As no such effects were noted in animals treated with 500 mg/kg bw/day, these effects were considered to be incidental and unrelated to treatment with the test item.
No such effects were noted in female animals treated with 500 mg/kg bw/day or in animals of either sex treated with 75 mg/kg bw/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The female animal treated with 250 mg/kg bw/day which was found dead on Day 51 did not show any abnormalities at necropsy.
There were no findings amongst surviving animals at necropsy that could be attributed to treatment with the test item.
The following findings were considered to be incidental and unrelated to treatment with the test item as they were generally isolated in nature and there were no histopathological correlates.
One male animal treated with 75 mg/kg bw/day exhibited a small and flaccid left testis with the corresponding epididymis also small.
One male animal treated with 250 mg/kg bw/day exhibited a fluid filled colon, multiple masses on the right epididymis, a mass on the right kidney, a mass on the prostate and multiple masses on urinary bladder, which was also enlarged.
One male and one female treated with 500 mg/kg bw/day exhibited a pale pituitary or an increased pelvic space in the kidney respectively.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Premature Decedents :
There was 1 premature decedent animal treated with 250 mg/kg bw/day which died during the bleeding procedure. No changes were noted at necropsy or histopathology which could be related to treatment with the test item.
Stomach:
Changes in the glandular region including ulceration, erosion and inflammatory cell infiltration were present in all males treated with 500 mg/kg bw/day.
Diffuse or focal hyperplasia of the non-glandular region, occasionally accompanied by inflammatory cell infiltration was present in 4/5 males and one female treated with 500 mg/kg bw/day.
The stomach findings were considered likely to be indicative of local irritation rather than demonstrating systemic toxicity, however is considered to be adverse in the animals affected.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis: Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 75, 250 or 500 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: 237 mg/kg bw/day of THEMAH
Key result
Critical effects observed:
no

Analytical verification

The results indicate that the prepared formulations were within ± 11% of the nominal concentration.

Conclusions:
The oral administration of Tris (2-hydroxyethyl) methylammonium hydroxide CAS 3366748-0 to rats by gavage, at dose levels of 75, 250 and 500 mg/kg bw/day, resulted in changes in the stomach of males and females treated with 500 mg/kg bw/day. This was considered to be indicative of local irritation rather than demonstrating systemic toxicity, however is considered to be adverse in the animals affected. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 500 mg/kg bw/day. A No Observed Adverse Effect Level (NOAEL) can be established at 500 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity as the effects noted are considered to be in relation to the irritant nature of the test item.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat

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:
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:
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:
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:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity - oral:

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 75, 250 and 500 mg/kg bw/day (equivalent to approximately 36, 119 and 237 mg/kg bw/day of THEMAH).  A control group of twelve males and twelve females was dosed with vehicle alone (Distilled water) over the same period.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 12 post partum.  Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.  Additionally, blood samples were taken at termination from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13 post partum, for thyroid hormone analysis; samples from adult males and Day 13 offspring were analyzed for Thyroxine (T4).

One female animal treated with 250 mg/kg bw/day was found dead on Day 51.  This occurred during the blood sampling procedure and was therefore considered unrelated to treatment with the test item. There were no further unscheduled deaths during the study. There were no clinical signs apparent that were considered to be related to systemic toxicity of the test item.Isolated occurrences of increased salivation were noted in animals of either sex treated with 500 mg/kg bw/day, occasional instances of noisy respiration were also noted in these animals.  Isolated occurrences of noisy respiration were noted in animals of either sex treated with 250 mg/kg bw/day. No clinical signs were apparent in any animal of either sex treated with 75 mg/kg bw/day. Signs of noisy respiration were also noted in one male and one female from the control group. Sporadic instances of noisy respiration were noted in one male and one female animal treated with 250 mg/kg bw/day and in four males and two female animals treated with 500 mg/kg bw/day and was noted a maximum of three times per animal during the treatment period.  One control male animal also exhibited noisy respiration on four separate occasions.

There were no treatment-related changes in the behavioral parameters in animals of either sex treated with 75 mg/kg bw/day. There were considered to be no treatment related changes in functional performance considered to be related to treatment at 75, 250 or 500 mg/kg bw/day. There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 75, 250 or 500 mg/kg bw/day. There were considered to be no adverse effects on body weight development in treated male animals during the study.

There were considered to be no effects on body weight development in treated females during maturation and no adverse effects during gestation or lactation. There was considered to be no effects in food consumption in any treated male animals.

There was considered to be no effects in food consumption in female animals during maturation and no adverse effects in food consumption in females during gestation or lactation. At 500 and 250 mg/kg bw/day, food conversion efficiency for males appeared inferior to control during the pre-pairing phase of the study.  At 75 mg/kg bw/day, food conversion efficiency for males appeared unaffected by treatment.

Food conversion efficiency for females during the pre-pairing phase of the study appeared unaffected by treatment at 75, 250 or 500 mg/kg bw/day. There were considered to be no effects on water consumption for any treated animal. There were no toxicologically significant effects detected in the hematological parameters examined. There were considered to be no toxicologically significant effects detected in the blood chemical parameters examined. The female animal treated with 250 mg/kg bw/day which was found dead on Day 51 did not show any abnormalities at necropsy.  There were no findings amongst surviving animals at necropsy that could be attributed to treatment with the test item.Evaluation of Thyroxine (T4) in adult males and offspring at Day 13 of age did not identify any obvious effect of treatment or indication of endocrine disruption at 75, 250 or 500 mg/kg bw/day. There were considered to be no toxicologically significant effects detected in the organ weights measured.

Histopathology results:

Premature Decedents: There was one premature decedent animal treated with 250 mg/kg bw/day which died during the bleeding procedure.  No changes were noted at necropsy or histopathology which could be related to treatment with the test item.

Stomach: Changes in the glandular region including ulceration, erosion and inflammatory cell infiltration were present in all males treated with 500 mg/kg bw/day.

Diffuse or focal hyperplasia of the non-glandular region, occasionally accompanied by inflammatory cell infiltration was present in 4/5 males and one female treated with 500 mg/kg bw/day. The oral administration of Tris (2-hydroxyethyl) methylammonium hydroxide (CAS 3366748 -0) to rats by gavage, at dose levels of 75, 250 and 500 mg/kg bw/day, resulted in changes in the stomach of males and females treated with 500 mg/kg bw/day. This was considered to be indicative of local irritation rather than demonstrating systemic toxicity. A No Observed Adverse Effect Level (NOAEL) can be established at 500 mg/kg bw/day for animals of either sex because the findings in general do not reflect true systemic toxicity as the effects noted are considered to be in relation to the irritant nature of the test item.

Repeated dose toxicity - 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 inhalation route of exposure.

Repeated dose toxicity - dermal:

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 route of exposure.

Justification for classification or non-classification

Based on the results in the combined repeated dose toxicity study with reproductive/developmental screening (according to OECD guideline 422), the test substance is not to be classified as STOT RE, according to CLP regulation.