Registration Dossier

Administrative data

Description of key information

In a 90d repeated dose toxicity study (OECD 408) with the test substance the NOAEL was 1.000 mg/kg/d.
    
    
    

g/d.

Repeated dose toxicity studies performed with on reaction partner (TMP) report NOAEL values of 100 and 200 mg/kg bw/d, and 0.3% in the diet, respectivley.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Older proprietary study, conducted by a reputable laboratory according to scientifically accepted methods, similar to current guidelines.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
The methods are comparable to OECD 408.
GLP compliance:
no
Remarks:
: study predates GLP
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
The animals were male and female weanling Wistar-derived albino rats from the test facility colony. They were assigned to treatment groups by body weight.
Food and water were provided ad libitum. The rats were housed in groups of 5 in screen bottom cages, in a room kept at a constant temperature of approximately 24°C.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Groups of rats were fed the stock diet (see additional information on materials and methods) with TMP added at levels of 0, 0.03, 0.1, 0.3 and 1.0%. The test compound was thoroughly mixed into the diet using a mechanical blender. The diets were freshly prepared once a fortnight and were stored at room temperature.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily - ad libitum in diet
Remarks:
Doses / Concentrations:
0, 0.03, 0.1, 0.3 and 1.0%
Basis:
nominal in diet
No. of animals per sex per dose:
Ten rats/sex/dose
Control animals:
yes, plain diet
Details on study design:
Rats were assigned to groups by body weight, and housed in groups of 5. There were 10 rats/sex/dose. Test diets were available ad libitum.
Positive control:
A positive control was not examined.
Observations and examinations performed and frequency:
Rats were observed for mortality, general condition and behaviour.
Individual body weights were recorded weekly. The food intake (and food efficiency) of each group was determined during the first 4 weeks, and during weeks 11 and 12.
Haematology parameters determined at weeks 4 and 12 were: haemaglobin content, packed cell volume, red blood cell count, total and differential white blood cell count.
Clinical chemistry parameters determined at study termination were: serum glutamic-pyruvic transaminase (SGPT), serum glutamic-oxaloacetic transaminase (SGOT), serum alkaline phosphatase (SAP), total serum protein, albumin and albumin-globulin ratio.
Urinalysis was conducted upon pooled urine samples of 10 males and 10 females of each group in week 13: appearance, pH, glucose, albumin, occult blood and ketones.
Sacrifice and pathology:
In week 14 all rats were killed by decapitation and examined macroscopically for pathological changes. The following organs were weighed: heart, kidney, liver, spleen, brain, testicle, ovary, thymus, pituitary, thyroid and adrenal gland. Samples of these organs and of a wide range of other organs were fixed in a 4% neutralised formaldehyde solution.
Detailed microscopic examination was performed on all male and female rats of the high dose and control groups. Haematoxylin-eosin stained paraffin sections of the organs weighed and also of the following organs were examined: lung, salivary glands, GI tract, trachea, skeletal muscle, aorta, exorbital lacrimal gland, axillary and mesenteric lymph nodes, pancreas, skin, urinary bladder, sternum with marrow, prostate, epididymis, coagulating gland, seminal vesicle, preputial gland, uterus, spinal cord and femoral nerve. Microscopic examination of rats fed lower doses was restricted to the liver and spleen.
Other examinations:
No other examinations were reported.
Statistics:
Wilcoxon test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
There were no mortalities during the study. No abnormalities of condition or behaviour were observed. There were no significant differences in body weight between the various groups, although at the three highest dose levels the body weights of the males were consistently lower than controls. Food consumption and food efficiency did not show consistent differences between groups.
In females fed 1.0% TMP, the haemoglobin contents were slightly lower than in the controls. The difference was only significant at week 4. Red blood cell counts and packed cell colume of females were decreased at the two highest dose levels at week 12. The blood smears showed normoblasts and white blood cell fragments in males and females at 1.0% in week 4. In week 12, normoblasts were observed in males at 1.0% and females at 0.3% and 1.0%. The differential counts did not reveal consistent differences between groups.
There were no distinct or significant differences in clinical chemistry parameters between the experimental groups and the controls.
There was no alteration in the urine which could be attributed to TMP.
Spleen weights were significantly increased at 1.0% in both sexes. The average relative weights of kidneys, liver and ovary were significantly increased in females at 1.0%. In males, pituitary weight was increased at 1.0%. There were other occasional significant differences, however there was no dose relationship and therefore the findings were considered incidental.
Moderately enlarged, dark spleens were seen in males and females of the 1.0% group at gross necropsy. No other changes were seen. Occasional lesions found at autopsy such as early signs of murine pneumonia and unilateral hydronephrosis, are regularly seen in rats of this strain, and were therefore not considered to be treatment related,
Histpathological examination revealed changes in the spleen and liver. Increased numbers of small lymphocytes, normoblasts and megakaryocytes were encountered in the red pulp of the spleen in the 1.0% group. The white pulp of the spleen was unremarkable. Normoblasts and an increased number of small lymphocytes were also present in the sinusoids of the livers of the 1.0% group. Slightly enlarged Kupffer cells containing yellowish brown pigment were seen in two females of the highest dose group. At lower dose levels, the spleen and liver did not differ from those of the controls.
There were no other changes that could be attributed to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
0.1 other: % in diet
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
haematology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.3 other: % in diet
System:
haematopoietic
Organ:
spleen
other: Packed cell volume and red blood cell counts decreased in females at 0.3 and 1.0%. Blood smears contained normoblasts and white blood cell fragments in both sexes at 1.0% and normoblasts in females at 0.3%
Treatment related:
yes
Dose response relationship:
yes

None

Conclusions:
The 90 day NOAEL was considered to be 0.1% in the diet, for male and female rats. The dose level is calculated to be equivalent to 100 mg/kg bw/d.
Executive summary:

The oral toxicity of trimethylolpropane (TMP 99) was determined in a 90 day feeding study with male and female rats. The test substance was fed at 0, 0.03, 0.1, 0.3 and 1.0% in stock diet to groups of 10 male and 10 female rats. Rats were sacrificed and subject to necropsy at the end of the feeding period.

General appearance and behaviour, body weight gain, food intake and efficiency, blood serum enzyme activities, serum protein content and urine composition were not affected by treatment. Haemoglobin content was decreased in 1.0% females, whilst packed cell volume and red blood cell counts were decreased in females at 0.3 and 1.0%. Blood smears contained normoblasts and white blood cell fragments in both sexes at 1.0% and normoblasts in females at 0.3%. The relative weight of the spleen was increased in both sexes. Liver, kidney and ovary weights were increased in 1.0% females. Pituitary weight was increased in males at 1.0%.

Gross examination at autopsy revealed enlarged dark spleens in both sexes at 1.0%. Histopathology revealed increased numbers of cells in the red pulp of the spleen, and in the liver Kupffer cells laden with pigment, sinusoids containing normoblasts and too many small lymphocytes were revealed in the 1.0% group. It was therefore concluded that the 90 day NOAEL of TMP 99 is 0.1% in the diet, for male and female rats.

Endpoint:
sub-chronic toxicity: oral
Remarks:
13 weeks plus 4 week recovery
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 6, 2019 - March 9, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 408 (July 2018), Test method B.26
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
TG July 2018
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)
Version / remarks:
TG 2008
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The animals were male and female Sprague-Dawley Crl:CD(SD) rats (60 males, 60 females). The animals were supplied by Charles River Italia S.p.A, Calco (Lecco), Italy.
Sex:
male/female
Details on test animals or test system and environmental conditions:
The rats were aged 27-29 days old on arrival, and weighed 75-99 g. They were allowed to acclimatise to the laboratory conditions for 18 days prior to dosing, during which time the health status of the animals was assessed by thorough observations. After arrival, on 24 of October 2019, the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. Body weights were in the range of 76.4-84.4 g for males and 66.2-72.9 g for females. A health check was then performed by a veterinarian.

The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C±2°C and 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages as indicated in the relevant SOP. Nesting material was provided inside suitable bedding bags and changed at least twice a week. Drinking water was supplied ad libitum to each cage via water bottles, except when overnight urine samples were collected in the last week of treatment from main phase groups. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei,4, 20019, Settimo Milanese (MI), Italy), with certified dietary levels of phytoestrogen (genistein less than 350μg/gram of diet), was offered ad libitum throughout the study,except in cases when water was restricted as mentioned above. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at the test facility. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

On the day of allocation (5 days prior to the start of treatment) all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the 4 groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed up to 3 of one sex per cage. The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery(Figure 1) to minimise possible environmental effects. No replacements occurred after the first dose was administered.

Procedures and facilities were compliant with the requirements of the Directive 2010/63/EUon the protection of animals used for scientific purposes. The national transposition of theDirective is defined inDecreto Legislativo 26/2014.The test facility is fully accredited by AAALAC. Aspects of the protocol concerning animal welfare have been approved by the animal-welfare body.
Route of administration:
oral: gavage
Details on route of administration:
The test item was dissolved in the vehicle and administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight. Preparations were made weekly (concentrations of 10,30, and 100 mg/mL) according to stability data obtained from a previous study (Study No. A3648).
Vehicle:
water
Remarks:
softened water (by reverse osmosis)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification available via a previous non-GLP stability study (ERBC Study no. A3648).
Duration of treatment / exposure:
13 consecutive weeks, followed by a recovery period of 4 weeks for 5 males and 5 females from groups 1 and 4. Animals in the main phase were dosed up until the day before necropsy.
Frequency of treatment:
All animals were dosed once a day, 7 days a week.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1; includes recovery 4 wk phase
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4; includes recovery 4 wk phase
No. of animals per sex per dose:
10 male and 10 female rats, control and high dose groups (group 1 & 4) included 5 additional animals per sex for 4 weeks of recovery
Control animals:
yes, concurrent vehicle
Details on study design:
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

The oral route was selected as it is a possible route of exposure of the test item in man and in agreement with authority’s decision.

Dose levels were selected in consultation with the Sponsor, based on information from aprevious study (non GLP ERBC Study No. E0419). In this study, the test item was admin-istered at the dose levels of 100, 300 and 1000 mg/kg/day, for two consecutive weeks and notreatment-related clinical signs nor differences in body weight, body weight gain and foodconsumption were recorded. At clinical pathology evaluations, non adverse alterations in some clinical chemistry parameters (such as urea, potassium and sodium) were recorded in females of the high dose group; however, the correlation with the test item was doubtful since no other changes were noted. No changes were observed in treated males. The post mortem macroscopic observations showed no relevant changes that could be related to treatment. For these above reasons, the high dose level tested of 1000 mg/kg/day was considered suitable for a toxicity study of longer duration.
Positive control:
None
Observations and examinations performed and frequency:
Throughout the study, all animals' clinical signs were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. From Day 1 to Day 71 of the treatment period, observations were performed approximately 2 hours postdose. Starting from Day 72, since clinical signs were noticed, observations were performed at approximately 30 minutes from administration.

Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly there after and just prior to necropsy.

The weight of food consumed by each cage of rats was recorded at weekly intervals followingallocation. The group mean daily intake per rat was calculated.
Sacrifice and pathology:
During the last week of treatment, individual overnight urine samples were collected fromall male and female animals from each main phase group under conditions of food and water deprivation.

At the same time interval, blood samples ofapproximately 0.5 mL for thyroid hormone determination (T3, T4, TSH) were collected fromthe sublingual vein, under light isoflurane anaesthesia and condition of food deprivation.

Prior to necropsy, blood samples were collected from all animals in main phase and recovery groups under isoflurane anaesthesia from the abdominal vena cava for haematology,coagulation and clinical chemistry, under conditions of food deprivation. Further blood and urine samples were taken under identical conditions at the end of the recovery period.

Animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia. All animals were subjected to necropsy, supervised by a pathologist. Requisite organs, according to TG 408, were weighed and the required tissue samples preserved in fixative. Necessary organ weights to body weights were calculated. Samples of all the tissues observed were fixed and preserved in 10% neutralbuffered formalin (except eyes, optic nerves, Harderian glands, testes and epididymideswhich were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examinations as outlines by TG 408 were performed on all animals in the control and high dose groups killed at the end of the 13 weeks of treatment and all specimens showing abnormalities in all main phase groups. Liver analysis was performed on all groups as possible treatment-related changes were observed. One cauda from one epididymis of each animal completing the scheduled test period in the high dose and the control group was taken for sperm count and evaluation of motility and morphology.
Other examinations:
Once before commencement of treatment and at least once per week during the study from the start of treatment, each animal was given a detailed clinical examination. Observations included gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypiesor bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes,occurrences of secretions and excretions. Once during Weeks 12 of treatment and once during Week 4 of recovery an evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptivestimuli), motor activity, and an assessment of grip strength was also performed.

Food consumption per cage was recorded weekly. The group mean daily intake per rat was calculated.

Both eyes of all animals were examined prior to the commencement of treatment by means of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropic-amide (Visumidriatic®, Visufarma, Rome, Italy). The eyes of all animals from high dose and control groups were re-examined during Week 13 of treatment.

At the end of the treatment and recovery periods, just prior to necropsy, vaginal smears were taken from all females.
Statistics:
Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was noticed starting from Week 11, in both genders treated at 1000 mg/kg/day. Almost all males (14 out of 15) and females (11 out of 15) showed salivation after 30 minutesfrom dosing. Some males treated with 300 mg/kg/day, at the end of Week 11 and/or beginning of Week 12 of the treatment period.Other signs, such as abrasion and scab on the tail, hairloss and tooth/teeth missing, were recorded during the study in single animals, with no correlation with the dose and were considered as minor clinical signs not related to the test item. Salivation was no longer observed during the recovery period and other signs recorded, such as swelling and hairloss, were considered as incidental and not relevant since the animals affected did not show any sign of toxicity during the treatment period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The only statistically significant increase found (+127%), that was not considered related to treatment, was the mean absolute body weight gain of males treated at 1000 mg/kg/day, on Day 22 of the recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Dosing phase:
Slight leucocytosis was recorded in males dosed at 300 and 1000 mg/kg/day. Compared with controls, changes were 31% and 57%, respectively, and involved mostly lymphocytes. Mean corpuscular volume was statistically significantly decreased in males dosed at 1000 mg/kg/day (3% below controls). Due to the minimal severity (values were withinthe range of historical data), this finding was considered to be not adverse. The other statistically significant differences between control and treated animals (haemoglobin in males, reticulocytes and lymphocytes in females) were recorded in animals dosed with 100 mg/kg/day, therefore they were considered to be unrelated to treatment.

Recovery phase:
In males, leucocytes were comparable with controls, confirming complete reversibility. In females, leucocytes were higher than controls. However, this was mostly due to the low control data; leucocytes values of the treated females were comparable with those recorded at end of the the dosing phase.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Many of the findings were mostly due to changes in one male dosed at 1000 mg/kg/day (animal no. A3649082): compared with mean control data, this animal showed increases of alanine aminotransferase (3.2 fold), aspartate aminotransferase (2.4 fold), total bilirubin (2fold), total cholesterol (2.1 fold, both HDL and LDL) and triglycerides (2.8 fold). Due to the minimal incidence, these findings cannot be conclusively attributed to treatment. Cholesterol and triglycerides changes were also recorded in a number of males dosed at 1000 mg/kg/day.The other statistically significant differences between control and treated animals (BUN/ureain both sexes, protein and albumin in males) were not dose-related, therefore they were considered to be incidental. All findings recorded at Week 13 showed a complete reversibility after 4 weeks of recovery.One treated male (no. A3649096) showed an increase of aspartate aminotransferase (2.4fold). Similar finding was not recorded at dosing phase (with the exception of animal no.A3649082), therefore this was considered to be incidental.

Alanine aminotransferase was still higher than controls in males (1.4 fold). However, the severity of this finding was insufficient to represent an hepatic disease, therefore thischange was considered to be not adverse.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean values of thyroid stimulating hormone were lower than mean control data in all malegroups. Changes were 21% to 53% with no clear dose-relation. Since low value was also recorded in one control male (no. A3649012), no dose-relation or statistically significance were achieved and no concurrent changes of the other hormones were recorded, the above finding was considered to be of no toxicological relevance.The statistically significant difference of thyroxine recorded between control and females dosed at 100 mg/kg/day (42% above controls) was not dose-related, therefore it was considered to be incidental.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant mild increase in mean liver weight was recorded in males treated at 300 mg/kg/day (relative weight only) and 1000 mg/kg/day (absolute and relative weights), compared to controls. This increase was dose-related (+10% and+24% in mid- and high dose groups for relative weights) and accompanied by corroborative histopathological changes, such as centrilobular hypertrophy. A statistically significant increase in mean relative kidneys weight (approximately +11% of relative weight) and in absolute and relative mean spleen weights (approximately +17% of relative weight) was recorded in males treated at 1000 mg/kg/day, when compared to controls. However, since these weight variations were not accompanied by corroborating histopathological changes in both kidneys and spleen, these were considered not toxicologically relevant. The minimal statistically significant increase (+11%), still observed in the liver of males previously dosed at 1000 mg/kg/day, was considered not toxicologically relevant since, at histomorphology, the liver was comparable to control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All observed changes were considered spontaneous and incidental, having a comparableincidence in control and treated groups and/or are characteristically seen in untreatedSprague Dawley SD rats of the same age. In particular, the mass observed at necropsy in the prostate of one high dose treated male (no. A3649082) corresponded microscopicallyto a benign prostatic adenoma in the ventral lobe and it was considered as a spontaneous pathology.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period, a slight but statistically significant increase in mean grip strength was recorded in females treated at 100 mg/kg/day (+16%). At the end of recovery, this parameter was found increased in males (+31%), and decreased in females (-22%) treated at 1000 mg/kg/day. No toxicological significance was attributed to these findings, since almost all values obtained were comparable to the other values recorded during the study and were there therefore considered to be incidental.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed treatment-related changes in the liver, consisted in centrilobular hepatocytic hypertrophy, morphologically represented by an enlarged size of the hepatocytes, of males treated at 1000mg/kg/day. This change was no longer observed at the end of the 4-week recovery period. This change correlated with the increased liver weight observed in mid- and high dose males and was most likely considered related to microsomal enzyme induction.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
All other reported findings, including the benign prostatic adenoma in the prostate ventrallobe of a single high dose male (no. A3649082), were considered spontaneous and incidental, having a comparable incidence in control and treated groups. In particular, the nephropathy observed was not considered treatment-related because it was observed in male rats of all groups, including control animals; the nephropathy was characterised by tubular degeneration/regeneration and/or necrosis and/or granular casts, in some cases associated with mononuclear cell infiltration, localised in the cortex and/or in the cortical/medullary junction.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The effects seen in males, slight leucocytosis, increased cholesterol and triglycerides levels and centrilobular hepatocytocytic hypertrophy in the liver, were treatment related, but reversible and therefore the not defined as adverse effects.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The slight leucocytosis and increased cholesterol and triglycerides and the histopathological effects (centrilobular hepatocytic hypertrophy) in males were considered to be related to treatment but, reversible and therefore not defined as adverse effects.
Key result
Critical effects observed:
no
Conclusions:
A repeated dose 90-day oral toxicity study with exposyre to Polyol TD by daily gavage in the rat was performed according to OECD guideline and GLP principles. No Observed Adverse Effect Level (NOAEL) for this study was 1000 mg/kg/day and the No Observed Effect Level(NOEL) was 100 mg/kg/day. There was slight leucocytosis in males treated at 300 and 1000 mg/kg/day, increased cholesterol and triglycerides in some males dosed at 1000 mg/kg/day and centrilobular hepatocytic hypertrophy in the liver of males dosed at 1000 mg/kg/day. The above findings were treatment related, but were no longer observed at the end of the recovery period and therefore were not defined as adverse effects.
Executive summary:

The toxicity of Polyol TD in rats after oral administration for 13 consecutive weeks and recovery from any treatment-related effects during a period of 4 weeks were investigated in accordance to OECD guideline 408 and GLP principles. Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 100, 300 and 1000 mg/kg body weight/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (softened water) and acted as a control. Five additional animals for each sex were included in the high dose and controlgroups for recovery assessment. The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, ophthalmoscopy, oestrous cycle, clinical pathology investigations, sperm analysis, terminal body weight, organweights, macroscopic observations and histopathological examination.

Signs of effects related to treatment were mainly observed in animals from the high dose group. Most of these effects were recorded at clinical pathology evaluations (slight leucocytosis inmales treated at 300 and 1000 mg/kg/day, with a dose-relation trend and increased cholesterol and triglycerides in some males dosed at 1000 mg/kg/day) and at histopathology(centrilobular hepatocytic hypertrophy in the liver of males dosed at 1000 mg/kg/day). These findings were considered to be related to treatment but, since they were no longer observed at the end of the recovery period, they were not defined as adverse effects.

Based on the above mentioned results, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 1000 mg/kg/day and the No Observed Effect Level(NOEL) was 100 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

Repeated dose oral toxicity:

Studies are available for the component TMP and therefore (by read-across) for the component DMP.

Waivers are proposed for repeated dermal and inhalation toxicity, in accordance with Column 2 of Annex VIII/IX of the REACH Regulation. The repeated dose toxicity of the substance has been adequately elucidated by the oral route, with additional testing proposed for CTF. Further testing by the addtional routes of exposure is not required.

TMP studies

The oral toxicity of trimethylolpropane (TMP 99) was determined in a 90 day feeding study with male and female rats (de Knecht-van Eekelen and van der Meulen, 1969). The test substance was fed at 0, 0.03, 0.1, 0.3 and 1.0% in stock diet to groups of 10 male and 10 female rats. Rats were sacrificed and subject to necropsy at the end of the feeding period. General appearance and behaviour, body weight gain, food intake and efficiency, blood serum enzyme activities, serum protein content and urine composition were not affected by treatment. Haemoglobin content was decreased in 1.0% females, whilst packed cell volume and red blood cell counts were decreased in females at 0.3 and 1.0%. Blood smears contained normoblasts and white blood cell fragments in both sexes at 1.0% and normoblasts in females at 0.3%. The relative weight of the spleen was increased in both sexes. Liver, kidney and ovary weights were increased in 1.0% females. Pituitary weight was increased in males at 1.0%. Gross examination at autopsy revealed enlarged dark spleens in both sexes at 1.0%. Histopathology revealed increased numbers of cells in the red pulp of the spleen, and in the liver Kupffer cells laden with pigment, sinusoids containing normoblasts and too many small lymphocytes were revealed in the 1.0% group. It was therefore concluded that the 90 day NOAEL of TMP 99 is 0.1% in the diet, for male and female rats. This dose level is calculated to be equivalent to 100 mg/kg bw/d, using default conversion factors.

A combined repeat dose and reproductive/developmental toxicity screening test was conducted in male and female Slc:SD rats (MHLW, 1994), according to GLP. Trimethylolpropane was administered daily to the rats by gavage, at doses of 0 (vehicle control; distilled water), 12.5, 50, 200, 800 mg/kg bw/day. The rats were dosed for 2 weeks prior to mating, during the mating period and pregnancy until day 4 of lactation (approximately 45 days). There were no deaths or abnormal clinical signs attributable to treatment. The body weights of males and females in the 800 mg/kg group were lower than those of the control group during the premating period. Food consumption was not affected by treatment. Haematology and clinical chemistry investigations did not reveal any treatment-related abnormalities. Absolute and relative liver weights were significantly elevated in the 800 mg/kg males, and increased but not significantly in the 800 mg/kg females. Necropsy revealed hypertrophy of the liver in 3 male 800 mg/kg rats. Histopathological examination revealed no definite morphological lesions. Slight basophilic alteration of the renal tubular epithelial cells was observed in 1 female receiving 50 mg/kg, in 2 females receiving 200 mg/kg, and in 5 females receiving 800 mg/kg. These changes were not unequivocally attributable to the test substance administration, because of their limited distribution and limited degree, and because similar lesions were observed in male rats of all groups including the controls. The NOAEL was therefore considered to be 200 mg/kg bw/d in both sexes.

The oral toxicity of trimethylolpropane (TMP) was studied in a range finding test (Wijnands & Feron, 1969). The substance was fed at 0, 0.3, 1.0, 2.0 and 4.0% in the diet to albino rats, for a period of 28 days.

The LOEL = ca. 965 mg/kg bw/day (1%). Reduced body weights and decreased food efficiency were observed at the 4% level in both sexes. Haematology analyses conducted on day 25 revealed reduced haemoglobin values at the 2% and 4% levels. Red blood cell counts were decreased dose-dependently in the 1%, 2% and 4% groups. White blood cell counts were increased in the 4% group. Enlargement of the spleen and liver was observed at necropsy in the 2% and 4% animals. Relative liver, kidney, heart and spleen weights were significantly and dose-dependently increased in the 1%, 2% and 4% groups. Histopathological examination revealed treatment-related abnormalities in the spleen and liver of rats in the 1%, 2% and 4% group. Slight abnormalities in the kidney were noted in the 2% and 4% males.

The 28 day NOAEL is considered to be 0.3% (ca. 275 mg/kg bw/day).

However, a 90 d repeated dose toxicity study with the Reaction mass “Polyol TD (Reaction mass of 2-ethylpropane-1,3-diol and5-ethyl-1,3-dioxane-5-methanol and propylidynetrimethanol)” did not reveal significant systemic toxicity. This study reported a NOAEL of 1000 mg/kg/d and a NOEL of 100 mg/kg/d. As soon as the remaining test results are available the DNEL and CSR will be corrected accordingly.

Justification for classification or non-classification

There is no evidence of marked toxicity or any effects at relevant dose levels (<100 mg/kg bw/d), therefore no classification is proposed for repeated dose toxicity according to Directive 67/548/EEC or Regulation (EC) No 1272/2008.