2,2-bis(hydroxymethyl)propionic acid

Endpoint summary

Description of key information

A modern 90-day rat study performed with the substance reports a NOAEL of 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2013 to 02 July 2014

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:

yes

Remarks:

: Several minor deviations from the protocol were identified. These deviations were considered not to have impact on the integrity or outcome of this study.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of the test material used in the study report: Bis-MPA TM
- Batch no.: 521152
- Purity: 98.0%
- Appearance: white, neutral crystalline powder
- Expiry date: 01 December 2013
- Storage conditions: ambient laboratory temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Edinburgh stock, Charles River UK Ltd, Margate, Kent, UK
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 188-237 g for males and 128-180 g for females
- Fasting period before study: No
- Housing: 2 or 3 per cage by sex in suspended polycarbonate cages with stainless steel grid tops, solid bottoms and a separate stainless steel food hopper
- Diet (e.g. ad libitum): Rat and Mouse (modified) No. 1 Diet SQC Expanded ad libitum
- Water (e.g. ad libitum): water taken from the public water supply ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24°C
- Humidity (%): 44-76%
- Air changes (per hr): minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From: 23 Apr 2013 To: 24 Jul 2013

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/v) methyl cellulose in water for irrigation

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 0, 20, 50, 100 mg/mL
- Amount of vehicle: 10 ml/kg
- Lot/batch no. : 021M0067V and G27R031
- Purity: 0.5% (w/v) methyl cellulose in water for irrigation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis from test item formulations on day 1, week 6 and week 12 for all groups. Concentration and homogeneity were analysed. Analyses were performed by reverse phase HPLC with ELSD Detection using a validated analytical procedure.

Duration of treatment / exposure:

91 days

Frequency of treatment:

single, daily oral gavage dose

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

(vehicle control)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose

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 agreed with the Sponsor following examination of a previous toxicity study in which animals received 200, 500 or 1000 Bis-MPA™ mg/kg bw/day for up to 28 days. In that study, 1000 mg/kg bw/day was considered to be well tolerated with no effects recorded on clinical observations, body weight or food consumption profiles, organ weights or macroscopic investigations and therefore a suitable high dose level for this 13 week toxicity study.

- Section schedule rationale (if not random): The animals were euthanised rotating across dose groups such that similar numbers of animals from each group, including controls were necropsied at similar times throughout the day.

Positive control:

Not relevant for this study type

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Animals were checked for viability. All animals were examined at frequent intervals throughout the day for signs of ill health or reaction to treatment, staring immediately post dose, with particular attention paid to the first hour after dosing.
- Posture/condition on first approach (animal undisturbed), checked for: Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convulsions, Biting (of cage components or self mutilating), Vocalisations, Piloerection.
Ease of removal from the cage.
Condition of the eyes, checking for: Pupillary function, Miosis, Mydriasis, Exophthalmos, Encrustation, Lacrimation.
Condition of the coat. Presence of salivation. Overall ease of handling.
- Observations in a Standardised Arena (2 Min Observation Period): Latency (time to first locomotory movement), Level of mobility, Rearing, Grooming, Urination/defecation, Arousal (level of alertness), Posture, tremor/convulsions, vocalisation, piloerection, Palpebral closure, Gait abnormalities, Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week
- Animals received a detailed clinical examination including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta

BODY WEIGHT: Yes
- Time schedule for examinations: once during the pretrial period and daily from the first day of dosing (Day 1) until the end of then observation period (reported weekly). Body weighs recorded immediately before dosing were classified as Day 0 body weights.

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each cage of animals was recorded once during pretrial, and weekly during the dosing period

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: Yes
- Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the pretrial and during Week 13
- Dose groups that were examined: The eyes of all animals (including extras) were examined during the pretrial period. All animals in the control and high dose group (1000 mg/kg bw/day) were also examined during Week 13 of treatment

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 92 - 93
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Red blood cell count, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin concentration, Mean cell haemoglobin, Reticulocytes, Reticulocyte count (absolute), Red blood cell distribution width, Platelets, White blood cell count, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Large unstained cells, coagulation.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 92 - 93
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Glucose, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatise, Creatine phosphokinase, Lactate dehydrogenase, Sodium, Potassium, Chloride, Total protein, Albumin, Globulin, Albumin/globulin ratio, Cholesterol, Creatinine, Total bilirubin, Calcium, Inorganic phosphate.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 87 - 88
- Metabolism cages used for collection of urine: Yes over a period of 16-18 hours
- Animals fasted: Yes, animal had access to water only while in the metabolism cages
- Parameters checked: Microscopic evaluation of spun deposit, Colour, Turbidity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Leukocytes, Blood pigments, Urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during pretrial and once during the treatment period (Week 13)
- Dose groups that were examined: all animals
- Battery of functions tested: Reaction to sudden sound, Reaction to touch on the rump with a blunt probe, Grip strength, Pain perception, Landing foot splay, Motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were subject to necropsy including unsceduled deaths
- All animals were euthanised by exposure to a rising concentration of carbon dioxide, weighed, and exsanguinated by severance of major blood vessels. Animals were not fasted before their scheduled necropsy.
- Complete necropsy examination: evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
- The following organs were weighed at necropsy for all scheduled euthanasia animals: Brain, Epididymis, Adrenal gland, Pituitary gland, Prostate gland, Thyroid gland, Heart, Kidney, Liver, Lung, Ovary, Spleen, Testis, Thymus, Uterus. Paired organs were weighed separately and reported together. Organs were weighed before fixation unless otherwise noted. Organ to body weight ratio/percentage (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes
- Representative samples of the following tissues were collected from all animals: Animal identification, Aorta artery, Bone marrow smear, Bone marrow from femur, Bone marrow from sternum, Femur bone, Sternum bone, Brain, Cervix, Epididymis, Eye, Adrenal gland, Harderian gland, Lacrimal gland, Mammary gland, Parathyroid gland, Pituitary gland, Prostate gland, Salivary gland, Seminal vesicle gland, Thyroid gland, Gross lesions/masses, Gut-associated lymphoid tissue, Heart, Kidney, Large intestine (caecum, colon, rectum), Larynx, Liver, Lung, Mandibular lymph node, Mesenteric lymph node, Skeletal muscle, Nasal cavity, Optic nerve, Sciatic nerve, Oesophagus, Ovary, Oviduct, Pancreas, Pharynx, Skin, Small intestine (duodenum, ileum, jejunum), Spinal cord, Spleen, Stomach, Testis, Thymus, Tongue, Trachea, Ureter, Urinary bladder, Uterus, Vagina

Other examinations:

None

Statistics:

Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females were analysed separately. Body weight, food consumption, selected functional observational battery and motor activitydata, haematology, coagulation, clinical chemistry and selected urinalysis data were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student’s t test; i.e. pairwise comparisons were made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student’s t test).
In circumstances were it was not possible to perform the F-Max test due to zero standard deviation in at least one group, the non-parametric ANOVA results were reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA as above as an exploratory analysis.
In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data were subjected to a rank transformation prior to analysis. Where it was not possible to perform the F-Max test due to the small sample size (i.e. less than 3 animals in any group), the untransformed parametric ANCOVA results are reported.

Clinical signs:

no effects observed

Description (incidence and severity):

Two unscheduled deaths were not attributable to treatment

Mortality:

no mortality observed

Description (incidence):

Two unscheduled deaths were not attributable to treatment

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinary pH was statistically significantly lower in both males and females that received 500 or 1000 mg/kg bw/day

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased absolute and relative liver weight in males at 1000 mg/kg bw/day

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were two unscheduled deaths during the course of this study. A 200 mg/kg bw/day female was euthanized on Day 35 due to an accidental transfer within the testing facility and another 200 mg/kg bw/day female was euthanized moribund on Day 29. The moribund condition of this animal was considered related to a gavage procedural error based on gross and microscopic findings in the thoracic cavity, lungs and thymus.


BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related differences in group mean body weight or body weight gain.


FOOD CONSUMPTION
There were no treatment-related differences in food consumption.


WATER CONSUMPTION
Visual inspection of the water bottles indicated no observable differences in water consumption.


OPHTHALMOSCOPIC EXAMINATION
There were no findings during ophthalmoscopic examinations that could be attributed to the test substance.


HAEMATOLOGY
There were no notable inter-group differences in haematological parameters.


CLINICAL CHEMISTRY
There were no notable inter-group differences in clinical chemistry parameters.


URINALYSIS
Urinary pH was statistically significantly lower in both males and females that received 500 or 1000 mg/kg bw/day.


NEUROBEHAVIOUR
All of the behaviours exhibited and observations were considered to be typical for rats of this age and strain on this type of study. There were no treatment-related differences in the quantitative functional observation parameters. There were no notable inter-group differences in motor activity.


ORGAN WEIGHTS
Absolute and relative liver weights were increased in the 1000 mg/kg bw/day Bis-MPA™ males.


GROSS PATHOLOGY
No treatment related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals.


HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals.


HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically significant effects of treatment were observed at the highest dose level.

Critical effects observed:

not specified

Table 1: Liver weights

Liver weight	M				F
	0	200	500	1000	0	200	500	1000
Absolute (g)	12.37	13.21	12.45	14.23**	8.35	8.00	8.45	8.26
Corrected (g)	12.90	12.65	12.59	14.12**	8.26	8.10	8.49	8.24
Relative (%)	3.350	3.279	3.266	3.654**	3.428	3.434	3.581	3.442

Conclusions:

In a subchronic repeated dose toxicity study conducted according to OECD 408, daily oral gavage administration of Bis-MPA to rats for at least 91 consecutive days was well tolerated and produced no toxicologically relevant effects on at dose levels up to and including 1000 mg/kg bw/day when compared to the vehicle control. The NOAEL for this study is therefore 1000 mg kg/bw/day.

Executive summary:

In a subchronic toxicity study (conducted according to OECD TG 408), Bis-MPA (purity 98.0%) was administered to 10 rats/sex/dose by gavage at dose levels of 0, 200, 500, or 1000 mg/kg bw/day. There were no compound related effects in mortality, clinical signs, body weight, food consumption, ophthalmoscopy, hematology, clinical chemistry, qualitative or quantitative functional observations or motor activity assessments, gross and histologic pathology. Two unscheduled deaths at 200 mg/kg bw/day in two females were due to a possible gavage procedural error and accidental transfer out of the animal room. Urinalysis findings were limited to reduced urine pH in both sexes at 500 and 1000 mg/kg bw/day. This finding is likely to reflect urinary excretion of the test material and is not considered to be of toxicological significance. Mean absolute and relative liver weights were significantly increased in males only at the highest dose level; this finding (in the absence of clinical chemistry or histopathological correlates) is considered to be an adaptive effect and not of toxicological significance. A NOAEL of Bis-MPA is 1000 mg/kg bw/day. This subchronic toxicity study in the rats is acceptable and satisfies the guideline requirement for a subchronic oral study (OPPTS 870.3100; OECD 408) in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A high-quality 90-day rat study is available for the substance

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Findings in a 90-day rat toxicity study performed with Bis-MPA (Dimethylolpropionic acid) were limited to reduced urinary pH in both sexes at dose levels of 500 and 1000 mg/kg bw/day and significantly increased absolute and relative liver weight in male rats at the highest dose level of 1000 mg/kg bw/day. Urine analysis findings are likley to reflect urinary excretion of the test substance and are therefore not considered to be of toxicological significance. The effect on liver weight in the absence of clinical chemistry or histopathological correlates is considered to be an adaptive effect and not of toxicological significance. Bis-MPA (Dimethylolpropionic acid) shows very low toxicity following repeated oral administration to the rat. A NOAEL of 1000 mg/kg bw/day is therefore determined for this study.

Justification for classification or non-classification

Based on the data available, no classification is required for Bis-MPA (Dimethylolpropionic acid) under the CLP Regulation (EC) No 1272/2008.