3-methylpentane-1,5-diol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 February 2019 to 23 March 2019 (experimental dates)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-methylpentane-1,5-diol

EC Number:

224-709-1

EC Name:

3-methylpentane-1,5-diol

Cas Number:

4457-71-0

Molecular formula:

C6H14O2

IUPAC Name:

3-methylpentane-1,5-diol

Specific details on test material used for the study:

Test Material: 3-methylpentane-1,5-diol
Source: Kuraray
Description: Clear colourless liquid
Lot/Batch No.: 93023
Purity: 99.5%
CAS No.: 4457-71-0
Stability of test compound: Confirmed stable for the duration of the study (expiry date: 10 July 2020)
Test substance:
- Name of test material (as cited in study report): 3-Methyl-1,5-Pentanediol
- Molecular weight: 118.20
- Physical state: Colorless liquid
- Analytical purity: 99.18%
- Lot/batch No.: 63136
- Expiration date of the lot/batch:
- Storage condition of test material: Nitrogen substituted, and stored sealed at room temperature in a dark place
- Other: Specific gravity: 0.97 (20°20); Boiling point: 270°C; Freezing point: Less than -50°C; Manufacturer: Kuraray Co.; Supplier: Ministry of Health and Welfare Environmental Health Bureau Planning Section Office for Environmental Chemicals Safety; Date of receipt: 1995-09-6;

Medium:
-Name: Japanese Pharmacopoeia water for injection (Otsuka Pharmaceutical Factory Co.,)
-Lot No. 5C82 and 5K81
-Storage: At room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

refer below

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Species: Rat
Strain: Wistar
Age at dosing: 7-8 wks
Weight at dosing: M: 197 – 240g; F: 154-191g
Source: Charles River, Germany
Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
Water: Municipal water, ad libitum (pH adjusted to pH 2-8)
Housing: Housed 5 animals/cage of the same sex

ENVIRONMENTAL CONDITIONS
Temperature: 22 ±3°C
Humidity: 55 ± 10%
Air changes: ca. 10/h
Photoperiod: 12 h light/dark

IN-LIFE DATES: 12 February 2019 to 23 March 2019 (experimental dates)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

animals dosed orally via gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test article was prepared at concentrations of 0, 20, 60, and 200 mg/mL. A separate study (Eurofins Munich Study No. 187545) concluded that test article concentrations of 15 and 250 mg/mL were stable for =10 days at room temperature, refrigerated (2-8°C) and frozen (-15 to -35°C). These samples were homogenous after 60 minutes (no stirring). The prepared test formulations were prepared and stored in conjunction with the previous stability data generated.

Verification and homogeneity of the diet preparation containing the test article were determined by the analysis of three samples (from upper, middle, lower strata) from each dose level prepared at the start and end of the dosing phase.

Acceptance criteria for concentration analysis:
-Mean concentration of test article formulation 90-110% of nominal

Stability analysis:
- Stability of test article had previously been verified.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/gp

Control animals:

yes, concurrent vehicle

Details on study design:

After an acclimatisation period of ca. 5 days, rats were assigned to groups by randomisation based on weight. The test article, 3 methylpentane-1,5-diol was administered once daily orally to groups of rats for a period of 90 d. Animals (10/sex/gp), were administered the test article orally via gavage at concentrations of 0, 100, 300, 1000 mg/kg bw/d, employing a dose volume of 5 mL/kg bw. Following 90 d of treatment animals were subjected to complete necropsy. Body weight and food consumption were measured at regular intervals. Functional observational battery were performed prior to dosing and during the last week of exposure. Clinical pathology evaluations (haematology) were performed with all surviving animals subjected to complete gross necropsy and full histopathology. Fertility parameters (sperm counts) were determined at necropsy.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

Observations:
Animals were inspected at least once daily for signs of toxicity and mortality. Detailed clinical examinations were conducted once before administration (on the day before the administration start day) and once a week during the administration period.

Body weights:
Animals were weighed at initiation of dosing and weekly during administration and at study termination.

Food consumption:
Determined by weighing food supplied and food that remained were calculated as time-weighted averages at weekly intervals.

Water consumption:
Not conducted

Ophthalmological examination:
Conducted on all animals before the first administration and during the final week of the treatment period.

Neurological functional examinations:
The following evaluations (measurements) were performed prior to initiation of dosing and at wk 12: approach response, auditory stimulation, proprioceptive response, grip strength, rearing, and motor activity.

Haematology and clinical chemistry:
Conducted on days 91. Animals were fasted overnight prior to blood sampling.
Haematology: red blood cell parameters (haematocrit (commonly termed PCV), haemoglobin concentration (Hb), mean haemoglobin concentration (MHC), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), erythrocyte count, platelet count, reticulocyte count), white blood cell parameters (total and differential (neutrophils, lymphocytes, eosinophils, basophils, monocytes) leukocyte count, large unstained cells), coagulation parameters (activated partial thromboplastin time (APTT), prothrombin time (PT)).

Clinical chemistry: electrolytes (sodium, potassium), kidney function test (creatinine, urea), glucose, liver function tests (albumi, alkaline phosphatase (ALP), alanine aminotransferase (ALT [commonly referred to as glutamic pyruvic transaminase (GPT)]), aspartate aminotransferase (AST [commonly referred to as glutamic oxaloacetic transaminase (GOT), gamma-glutamyl transferase (G-GT)]), total bilirubin (T.Bili), total bile acids (TBA), total protein (TP), lipid profile (total triglyceride, total cholesterol, LDL, HDL), thyroid hormones (T3, T4, TSH).

Toxicokinetics:
Blood samples from 5 animals/sex/gp were taken at 30 minutes and 2 h post dosing in week 1, 4 and 13 to assess for possible systemic exposure and accumulation.

Urinalysis:
Conducted on day 91. The following urinary parameters were measured: specific gravity, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, leukocyte, nitrite

Organ weights:
Adrenal glands, brain, epididymides, heart, kidney, liver, ovary, pituitary gland, prostate (+seminal vesicles, coagulating gland), spleen, thymus, thyroid (+parathyroid), testis, uterus (+cervix).

Sacrifice and pathology:

Conducted on day 91. Gross pathological examination was performed on all animals and included examination of the external surface, all orifices and associated tissues.
The following tissues were preserved in 4% neutral buffered formalin for subsequent histopathological examination (with the exception of eyes, testes and epididymides which were fixed in Modified Davidson's fixative, then transferred to 70% ethanol) and performed on control and high dose group animals:
Accessory sex glands (M: epididymides, prostate (ventral and dorsolateral), seminal vesicle (coagulating gland), testes; F: ovary, uterus +cervix, vagina), cardiovascular/haematological system (aorta, bone (sternum for marrow), heart, lymph nodes (mesenteric, auxiliary), spleen, thymus), gastrointestinal tract (oesophagus, tongue, stomach, intestine (caecum, colon, duodenum, Peyer's patches, ileum, jejunum, rectum), liver, pancreas, salivary glands (submaxillary, sublingual, parotids'), neurological (brain (cerebellum, cerebrum, midbrain), eyes (+optic nerve, Harderian gland), sciatic nerve, spinal cord (cervical, thoracic, lumbar), respiratory system (trachea, lung), urogenital system (kidneys, urinary bladder), other (skeletal muscle, skin, all gross lesions and masses)
Other endocrine producing/sensitive glands (adrenals, mammary gland, pituitary, thyroid (+parathyroid))

Other examinations:

Spermatogenesis:
At necropsy, the left epididymis and left testis were separated and used for the evaluation of sperm parameters. Epididymal sperm motility and testicular sperm counts were undertaken for all males.

Neurohistopathology: No specific neurohistopathology with specific fixatives were performed in addition to the standard histopathology undertaken on neuronal tissues.

Statistics:

Body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were undertaken for each gender by comparing values of dosed animals with the respective control group animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related effects observed, with effects occurring sporadically, nor dose related and not reflective in both genders.

Mortality:

no mortality observed

Description (incidence):

All animals survived to scheduled necropsy from both sexes

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and weight gain were unaffected in either males or females treated up to 1000 mg/kg bw/day over the entire administration period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related changes in food consumption in either sex

Food efficiency:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in food efficiency in either sex

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

not examined

Description (incidence and severity):

n/a

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment related effects observed in either sex.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Haemoglobin was significantly elevated in mid and high dose group.
Neutrophils counts were significantly decreased in the high dose group
Basophil counts were significantly increased in mid dose group
APTT was significantly longer in high dose group

Females:
Prothrombin time in mid and high dose groups and platelet count in the mid dose gp were significantly increased

These changes were not deemed treatment related for the following reasons:
- Changes in the respective parameters, whilst achieving statistical significance were within the historical control range of the individual values reported in the conducting laboratory.
- The changes observed were not replicated in each sex.
- The effects observed were not dose related.
- In terms of coagulation parameters, these values in the treated groups were comparable to controls, with the statistically significant increases observed set against a low concurrent control value.
- In terms of the neutrophil and basophil counts, no other change in WBC parameters were observed, with no histopathological changes observed in associated tissues.
Refer to Table 7.8.2/02-4

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
AST was significantly increased in the high dose group
K was significantly increased in the high dose group

Females:
Creatinine was significantly lower in the high dose group
Total bile acids were significantly higher in the high dose group

These changes were not deemed treatment related for the following reasons:
- Changes in the respective parameters, whilst achieving statistical significance were within the historical control range of the individual values reported in the conducting laboratory.
- The changes observed were not replicated in each sex.
- In terms of coagulation parameters, these values in the treated groups were comparable to controls, with the statistically significant increases observed set against a low concurrent control value.
- In the absence of any histopathological findings to conclude hepatocellular injury, these effects were deemed not test article related.
- For total bile acids, there were no changes in either ALT, AST with no observed effect on hepatic function.
Refer to Table 7.8.2/02-5

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment related effects observed in either sex.

Behaviour (functional findings):

not examined

Description (incidence and severity):

n/a

Immunological findings:

not examined

Description (incidence and severity):

n/a

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males: a statistically significant increase in relative (to body weight) kidney weight was observed in the high dose group.
Females: a statistically significant increase in absolute kidney weight and relative (to body and to brain) weight was seen for the high dose group compared to the respective controls. In female animals a statistically significant higher absolute liver weight, and relative (to body and to brain) weight was seen for the high dose group compared to the respective control. Increase in kidney weight relative to brain weight was also seen at the mid dose group.
- The values for both organs were within the respective historical control ranges
- No histopathological changes were observed in the respective tissues.
- The effect in the liver were not replicated in males.
No other significant changes in organ weights (including thyroid) were observed.

Refer to Table 7.8.2/02-7

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were noted in any animal in the control or test article groups in either sex.

Neuropathological findings:

not examined

Description (incidence and severity):

n/a

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test article related effects were observed in either sex, with changes deemed representative of normal background variability within the Wistar rat at the conducting laboratory (refer to Table 7.5.2/02-9).
There was no histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix, and vagina. In addition, in the investigated testes no treatment-related effects on the testicular histopathology were observed and the histological appearance reflected the animal physiology.
Thymus - minimal to slight multifocal hemorrhage within the thymus and moderate multifocal hemorrhages in the connective tissue adjacent to the thymus were observed in several control and treated animals. Further, minimal multifocal hemosiderin deposits mostly associated with the above mentioned hemorrhages were also observed in some control and high dose animals at incidences comparable to published data (McInnes, 2012). In the absence of additional thymic changes (e.g. necrosis, inflammation), all the above mentioned changes in the thymus were considered most likely incidental were deemed to be not test item related [Firth, 2000, McInnes, 2012, 2017; NTP. Sefanski et al 1990).

Mesenteric lymph nodes - minimal to slight multifocal hemorrhages were observed in several animals from the control and high dose group. Further, slight hemosiderin deposits within macrophages was observed in one female from the high dose group only. Haemorrhages and bron pigment deposition (e.g. haemosiderin, ceroid lipofusin) are background lesions commonly observed in lymph nodes of rats (McInnes, 2000, 2017, Stefanski et al 1990). Furthermore, in this present study the incidence and severity of the above mentioned changes was similar to the published data and all observed in the mesenteric lymph nodes changes were considered to be most likely incidental and deemed to be not test item related (McInnes, 2012, 2017).

References:
Firth, C.H., Ward, J.M., Chandra, M., Losco, P.E. 2000. Non-proliferative lesions of the hematopoietic system in rats. In: HL-1 guides for toxicologic pathology. STP/ARP/AFIP, Washington DC
McInnes, E.F. 2012. Wistar and Sprague Dawley rats. In: McInnes, E.F. and Mann, P. (eds. Background lesions in laboratory animals. A colour Atlas. Saunders Elsevier, Toronto, pp. 17-36
McInnes, E.F. 2017. Common spontaneous and background lesions in laboratory animals. Principles and Practices of Laboratory Animal pathology for Study Personnel. Wiley Blackwell, West Sussex, pp. 62-69
NTP: https://ntp.niehs.nih.gov/nnl/immune/thymus/hemorr/index.htm
Stefanski, S.A, Elwell, M.R., Stromberg, P.E. 1990. Spleen, lymph nodes and thymus. In: Pathology of the Fischer Rat: Reference and Atlas (Boorman G.A, Eustis, S.L., Elwell, M.R., Montgomery, C.A., MacKenzie, W.F. eds). Academic Press, San Diego

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

n/a

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones:
T3 was significantly elevated in females at both the mid and high dose groups, exceeding, but were not deemed biologically relevant for the following reasons:
- Due to the lack of historical control data, in this instance, the biological relevance assessment should be made against the concurrent control group which had an individual animal range of 0.73 – 2.01 ng/mL. In the treated groups, 2/10, 5/10 and 5/10 animals exceeded the upper concurrent vehicle control range for the low, mid and high dose groups respectively. No dose response relationship was evident at the individual animal level. These data are consistent with open literature data drawn on a greater number of studies.
- The changes observed were not replicated in each sex.
- No associated histopathological changes in the liver, thyroid or pituitary gland were observed.
- Both T4 and TSH levels were comparable with the concurrent vehicle control.
Refer to Table 7.8.2/02-6

Spermatogenesis:
There were treatment related effects observed in mean testis weights, mean sperm count, mean sperm motility for all dose groups in the treatment period. Mean total number of abnormal and normal sperms/findings and sperm morphology for high dose group animals were comparable with the vehicle control.
Refer to Table 7.8.2/02-8

Effect levels

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 7.5.1/02-1: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: dose formulation analysis

Parameters	Dose (mg/mL)
	20	60	200
Week 1	90.8	90.5	98.6
Week 5	99.1	98.2	105.1
Week 9	97.8	98.6	105.7
Week 12	97.0	91.4	96.

Table 7.5.1/02-2: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: Functional observations

Parameters		M (mg/kg bw/d)				F (mg/kg bw/d)
	Wk	0	100	300	1000	0	100	300	1000
Open field grooming	-1 13	0.00 0.40	0.10 0.00^**	0.00 0.00^**	0.00 0.00^**	0.20 0.00	0.10 0.00	0.00 0.00	0.00 0.00
Response to handling	-1 13	4.20 4.00	4.30 3.80	4.00 3.70	4.10 3.00^**	4.60 4.20	4.40 4.20	4.20 4.10	4.40 4.00
Spontaneous activity	-1 13	4.00 4.00	4.00 4.00	4.00 3.90	3.90 3.50^**	4.00 4.00	4.00 4.00	4.00 4.00	4.00 4.00
Rearing supported	-1 13	2.70 4.00	4.50 4.50	3.80 4.10	2.80 2.20^*	5.60 4.10	4.60 5.10	5.10 6.00	4.20 4.90
Temperature (°C)	-1 13	37.99 38.440	38.220 37.700^*	38.330 37.870^*	38.040 38.240	38.130 38.990	38.310 39.00	38.440 38.650	38.300 38.940

Table 7.5.1/02-3: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with3-methylpentane-1,5-diol: body weight effects

Parameters		M (mg/kg bw/d)				F (mg/kg bw/d)
		0	100	300	1000	0	100	300	1000
Body wt (g)	Wk 1 Wk 7 Wk 13	217.30 350.60 394.00	219.30 348.10 387.90	217.50 344.50 388.10	216.10 335.10 372.80	174.50 216.80 227.80	171.40 218.80 232.20	170.70 213.10 228.70	171.60 214.70 225.50
Body wt gain (g)	Wk 0-13	176.70	168.60	170.60	156.70	53.30	60.80	58.00	53.90

Table 7.5.1/02-4: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with3-methylpentane-1,5-diol: selected haematological parameters

Parameters	M (mg/kg bw/d)					F (mg/kg bw/d)
	0	100	300	1000		0	100	300	1000
Hb (g/dL)	14.744	15.300	15.544^*	15.562^*		14.040	14.450	14.530	14.640
WBC (10^9/L)	3.1967	4.2089	5.2000	4.3212		2.0260	2.2060	2.8480	2.2700
Eosinophils (%)	0.5444	0.4222	0.2222	0.9250		0.6800	0.3900	0.3800	0.2600
Lymphocytes (%)	73.744	79.911	78.789	80.112		79.120	79.800	81.730	85.410
Neutrophils (%)	23.522	17.578	19.022	16.275^*		17.870	16.980	15.450	11.970
Monocytes (%)	1.800	1.678	1.444	2.238		1.940	2.400	1.990	2.050
Basophils (%)	0.0778	0.1333	0.1667^*	0.1625		0.0600	0.1000	0.1200	0.1000
PT (sec)	22.644	22.600	23.080	23.189		24.480	25.300	25.680^**	26.470^***
APTT (sec)	12.810	13.370	13.260	14.422^**		11.610	11.840	12.340	12.530
Parameters	Laboratory historical control data (2011 – 2019) Wistar rat
	n	Observed range	Mean ±SD	Mean -/+2SD		n	Observed range	Mean ±SD	Mean -/+2SD
Hb (g/dL)	255	13.7 – 18.5	16.2 ±0.9	14.5 - 17.9		250	12.6 – 17.5	15.2 ±0.9	13.4 – 17.0
WBC (10^9/L)	256	1.5 – 13.8	4.7 ±1.5	1.6 – 7.8		250	0.3 – 6.8	2.6 ±1.2	0.3 – 5.0
Eosinophils (%)	256	0.4 – 1.9	0.6 ±0.4	-0.1 – 1.3		250	0.0 – 6.9	0.6 ±0.7	-0.7 – 2.0
Lymphocytes (%)	256	27.3 – 88.8	60.3 – 90.2	75.3 ±7.5		250	4.3 – 89.4	77.4 ±10.5	56.3 – 98.4
Neutrophils (%)	256	8.0 – 69.4	20.9 ±7.3	1.6 – 7.8		250	7.2 – 82.0	18.9 ±9.8	-0.8 – 38.6
Monocytes (%)	256	0.7 – 7.2	2.7 ±1.4	0.5 – 74.9		250	0.7 – 13.5	2.6 ±1.4	-0.3 – 5.5
Basophils (%)	256	0.0 – 0.5	0.2 ±0.1	0.0 – 0.4		250	0.0 – 0.9	0.1 ±0.1	-0.1 – 0.3
PT (sec)	200	12.5 – 26.0	21.6 ±1.8	17.9 – 25.3		188	13.9 – 39.7	22.9 ±2.3	18.3 – 27.4
APTT (sec)	201	8.4 – 30.1	14.1 ±3.2	7.7 – 20.6		189	8.7 – 25.5	13.6 ±2.8	8.0 – 19.2
*p=0.05, **p=0.01; ***p=0.001 Hb: haemoglobin WBC: white blood cell count APTT: activated partial thromboplastin time					PT: prothrombin

Table 7.5.1/02-5: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with3-methylpentane-1,5-diol: selected clinical chemistry parameters

Parameters	M (mg/kg bw/d)					F (mg/kg bw/d)
	0	100	300	1000		0	100	300	1000
ALT (U/L)	31.080	34.970	35.370	41.720		27.090	31.750	26.970	31.030
AST (U/L)	88.620	88.270	98.720	110.060^*		84.510	80.860	78.080	78.920
ALP (U/L)	88.0270	82.3250	79.7870	79.4200		56.7970	43.6820	48.2260	46.2940
¿GT (U/L)	0.2450	0.2360	2.6957	0.3540		0.7670	0.4275	0.4611	0.4688
Creat. (µmol/L)	22.50	21.10	29.90	23.30		26.60	26.70	23.30	21.90^**
K (mmol/L)	4.4820	4.2490	5.0740	7.1070^***		4.0130	3.9330	4.7300	4.9610
T.bili (µmol/L)	1.860	1.970	2.110	2.250		2.050	2.080	2.490^*	2.340
TBA (µmol/L)	29.5080	24.9550	32.0000	34.0210		19.1920	28.7000	25.8670	37.0730^*
Parameters	Laboratory historical control data (2011 – 2019) Wistar rat
	n	Observed range	Mean ±SD	Mean -/+2SD		n	Observed range	Mean ±SD	Mean -/+2SD
ALT (U/L)	214	8.1 – 107.9	35.1 ±12.3	10.6 – 59.6		201	3.5 – 84.7	28.5 ±10.3	7.9 – 49.1
AST (U/L)	115	31.3 – 159.8	88.6 ±24.4	39.6 – 137.7		201	12.6 – 211.8	77.8 ±25.3	27.1 – 128.4
ALP (U/L)	214	13.6 – 337.0	115.6 ±51.5	12.7 – 218.6		201	6.6 – 258.0	65.2 ±42.6	-20.1 – 150.4
gGT (U/L)	20	0.0 – 3.7	0.5 ±0.8	-1.1 – 2.1		23	0.0 – 2.3	0.6 ±0.5	-0.5 – 1.6
Creat. (µmol/L)	214	0.0 – 103.0	31.0 ±12.4	6.3 – 55.8		199	2.0 – 103.0	31.1 ±10.7	9.6 – 52.5
K (mmol/L)	214	1.4 – 8.7	3.9 ±0.8	-0.6 – 76.1		201	1.0 – 9.0	3.5 ±0.9	1.7 – 5.3
T.bili (µmol/L)	204	0.9 – 3.7	2.3 ±0.4	1.4 – 3.2		191	1.1 – 5.7	2.7 ±0.7	1.4 – 4.1
TBA (µmol/L)	143	2.1 – 110.0	37.8 ±0.4	-0.6 – 76.1		133	5.4 – 162.7	30.9 ±25.2	-19.5 – 81.4
*p=0.05; **p=0.01; ***p=0.001 ALT: alanine aminotransferase AST: aspartate transaminase ALP: alanine phosphatase					gGT: gamma glutamyl transferase Creat.: creatinine K: potassium T.bili.: total bilirubin TBA: total bile acids

Table 7.5.1/02-6: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with3-methylpentane-1,5-diol: thyroid hormones

Parameters	M (mg/kg bw/d)					F (mg/kg bw/d)
	0	100	300	1000		0	100	300	1000
T3 (ng/mL) [individual animal range]	1.97 [1.59 – 2.50]	2.04 [1.52 – 2.50]	1.68 [0.76 – 2.68]	2.17 [1.66 – 2.50]		1.43 [0.73 – 2.01]	1.63 [0.89 – 2.25]	1.98^* [1.30 – 2.48]	2.04^* [1.33 – 2.91]
T4 (ng/mL) [individual animal range]	55.56 [41.25 - 68.68]	57.92 [47.70 – 78.47]	50.61 [36.44 – 57.80]	55.87 [45.37 – 65.65]		38.58 [30.22 – 59.35]	38.35 [27.97 – 60.13]	41.28 [31.85 – 53.29]	45.63 [33.79 – 53.76]
TSH (ng/mL) [individual animal range]	0.57 [0.28 – 0.99]	0.60 [0.14 – 1.01]	0.67 [0.22 - 1.23]	0.96 [0.53 - 1.88]		0.63 [0.24 – 1.55]	0.79 [0.26 – 2.10]	0.75 [0.24 – 1.69]	0.96 [0.30 - 1.93]
*p=0.05; T3: triiodothyronine T4: thyroxine TSH: thyroid stimulating hormone

Table 7.5.1/02-7: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: selected organ weights

Parameters		M (mg/kg bw/d)					F (mg/kg bw/d)
		0	100	300	1000		0	100	300	1000
Terminal bwt (g)		380.20	373.90	370.80	358.30		222.40	225.10	223.60	218.40
Liver	Abs (g) Rel. (g%^b wt) (g%^br wt)	9.1128 2.3968 446.57	9.1358 2.4395 435.95	8.9011 2.4010 433.61	9.1312 2.5519 448.39		5..5761 2.171 284.79	5.6454 2.5110 294.70	5.8836 2.6347 298.81	6.1847^* 2.8279^** 320.833^**
Kidney	Abs (g) Rel. (g%^b wt) (g%^br wt)	2.1523 0.5674 105.41	2.2865 0.6123 109.19	2.2975 0.6197 111.77	2.3699 0.6631^*** 116.21		1.5066 0.6803 76.88	1.5795 0.7027 82.39	1.6301 0.7302 82.88^*	1.7181^*** 0.7868^** 89.11^***
Thyroid/parathyroid	Abs (g) Rel. (g%^b wt) (g%^br wt)	0.0334 0.0088 1.63	0.0335 0.0090 1.61	0.0312 0.0084 1.52	0.0315 0.0087 1.54		0.0245 0.0110 1.25	0.0273 0.0121 1.42	0.0263 0.0118 1.33	0.0266 0.0121 1.38
Testes	Abs (g) Rel. (g%^b wt) (g%^br wt)	3.6115 0.9546 176.98	3.5371 0.9493 169.59	3.6955 0.9985 179.79	3.7218 1.0431 182.48		-	-	-	-
Epididymides	Abs (g) Rel. (g%^b wt) (g%^br wt)	1.3267 0.3525 65.04	1.2452 0.3340 59.61	1.2710 0.3434 61.84	1.3703 0.3839 67.19		-	-	-	-
Ovaries	Abs (g) Rel. (g%^b wt) (g%^br wt)	-	-	-	-		0.1121 0.0503 5.71	0.1094 0.0487 5.71	0.1107 0.0496 5.62	0.1055 0.0484 5.48
Uterus (+cervix)	Abs (g) Rel. (g%^b wt) (g%^br wt)	-	-	-	-		0.7100 0.3236 39.39	0.7667 0.3405 40.12	1.0725 0.4849 54.30	0.6480 0.2976 33.71
Parameters (absolute weights [g])		Laboratory historical control data (2011 – 2019) Wistar rat
		n	Observed range	Mean ±SD	Mean -/+2SD		n	Observed range	Mean ±SD	Mean -/+2SD
Liver		130	6.92 – 12.77	9.12 ±1.14	6.83 – 11.40		130	2.47 – 8.85	5.73 ±1.05	3.63 – 7.83
Kidney		130	1.71 – 3.12	2.33 ±0.26	1.82 – 2.84		130	0.67 – 1.94	1.47 ±0.25	0.97 – 1.96
Thyroid/ parathyroid		89	0.0098 – 0.0790	0.0257 ±0.0088	0.0080 – 0.0433		90	0.0068 – 0.0419	0.0188 ±0.0071	0.0046 – 0.0329
Testes		134	2.34 – 4.47	3.69 ±0.32	3.05 – 4.33		-	-	-	-
Epididymides		134	0.77 – 1.72	1.37 ±0.15	1.07 - 1.67		-	-	-	-
Ovaries		-	-	-	-		135	0.05 – 0.17	0.10 ±0.02	0.05 – 0.15
Uterus (+cervix)		-	-	-	-		134	0.22 – 1.82	0.82 ±0.33	0.17 – 1.48
*p=0.05; **p=0.01; ***p=0.01 Abs.: absolute						Rel (g%^b wt): relative to body weight Rel (g%^br wt): relative to brain weight

Table 7.5.1/02-8: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: spermatogenesis parameters

Parameters	Dose group (mg/kg bw/d)
	0	100	300	1000
Mean testes weight
Testes	1.855	1.790	1.864	1.883
Tunica albuginea	0.128	0.113	0.128	0.122
Calculated wt of parenchyma	1.727	1.677	1.736	1.762
Mean sperm motility (%)
Motile count	83.1	85.8	84.2	84.5
Static count	16.9	14.2	15.8	15.5
Rapid	67.3	69.9	66.7	68.4
Testicular sperm count (10^6/g)	105.2	91.0	100.0	97.4
Mean sperm morphology
Total no. of normal sperm/findings	193.20	-	-	192.50
Total no. of abnormal sperm/findings	6.80	-	-	7.50
% normal	96.60	-	-	96.25
% abnormal	3.40	-	-	3.75

Table 7.5.1/02-8: Overview of sub-chronic toxicity study in rats treated orally (via gavage) with 3-methylpentane-1,5-diol: selected histopathology data Parameters M (mg/kg bw/d) F (mg/kg bw/d) 0 100 300 1000 0 100 300 1000 HEART - Cart. metaplasia - Haem. deposits - Mono. infiltr 3/10 0/10 0/10 - - - - - - 2/10 0/10 0/10 2/10 1/10 1/10 - - - - - - 1/10 0/10 1/10 TRACHEA - Gland. ectasia - Mixed cell infiltr 4/10 0/10 - - - - 8/10 0/10 5/10 1/10 - - - - 5/10 0/10 LIVER - Haem. deposits - Necrosis - Fibrosis - Mono. Infiltr - Granuloma - Haematopoiesis - Vacuolation 0/10 0/10 0/10 0/10 9/10 1/10 0/10 1/1 1/1 1/1 1/1 0/1 0/1 0/1 - - - - - - - 0/10 0/10 0/10 0/10 7/10 0/10 0/10 0/10 0/10 0/10 0/10 8/10 0/10 0/10 - - - - - - - - - - - - - - 0/10 0/10 0/10 0/10 5/10 0/10 0/10 SPLEEN - Haem. deposits - Incr. haemop. 7/10 10/10 - - - - 9/10 8/10 10/10 10/10 - - - - 10/10 10/10 MESENTERIC LYMPH NODES - Haemorrhage - Histiocytosis - Haem. deposits - Lymphoid depl. 6/10 1/10 0/10 1/10 - - - - - - - - 3/10 0/10 0/10 2/10 2/10 0/10 0/10 1/10 - - - - - - - - 7/10 0/10 1/10 0/10 KIDNEYS - Hyal. droplets - Mineralisation - Mono. Infiltr. - Pelvic dilation - Tub. s. dilation 10/10 3/10 2/10 2/10 4/10 - - - - - - - - - - 5/10 0/10 0/10 3/10 4/10 0/10 3/10 0/10 1/10 1/10 - - - - - - - - - - 0/10 2/10 0/10 2/10 2/10 STOMACH - Hyperkeratosis - Mix. cell infiltr. - Mono. infiltr. - Squ. hyperlasia 2/10 1/10 1/10 2/10 - - - - - - - - 0/10 1/10 1/10 0/10 3/10 1/10 0/10 3/10 - - - - - - - - 1/10 0/10 0/10 1/10 LUNG - Haem. deposits - Alve. histiocyt. - Mixed cell infiltr - Mononuc. Infiltr - Vasc. minerlisa. 0/10 5/10 1/10 2/10 3/10 1/1 0/1 0/1 0/1 0/1 - - - - - - - - - - 1/10 4/10 1/10 0/10 4/10 - - - - - - - - - - 0/10 8/10 1/10 0/10 9/10 PAROTID GLANDS - Mononuc.Infiltr 0/10 - - 0/10 1/10 - - 0/10 THYMUS - Haemorrhage - Haem. deposits 0/10 0/10 2/2 0/2 3/3 0/3 0/10 0/10 2/10 2/10 1/1 0/1 1/2 0/2 5/10 4/10 EPIDIDYMIDES - Mononuc.infiltr - Pigmen. macrop. 1/10 1/10 - - - - 2/10 2/10 - - - - AXILLARY LYMPH NODES - Haem. deposits - Haemorrhage - Lymphoid depl. 0/10 1/10 2/10 - - - - - - 0/10 0/10 1/10 1/10 1/10 3/10 - - - - - - 0/10 0/10 2/10 ADRENAL CORTICLES - Vacuolisation 3/10 - - 3/10 1/10 - - 0/10 VAGINA: OESTROUS CYCLE - Proestrus - Oestrus - Diestrus - - - - 1/9 1/9 7/9 - - - - - - 0/10 2/10 8/10 Cart. metaplasia: cartilage metaphase Haem. deposits: Haemosiderin deposits Mono. Infiltr.: mononuclear infiltrates Gland. ectasia : Glandular ectasia Mixed cell infiltr: mixed cell infiltrates Incr. haematop.: increased haemopoiesis Lymphoid depl.: lymphoid depletion Hyal. droplets: hyaline droplets Tub. s. dilation: tubular simple dilation Mix. cell infiltr : mixed cell infiltrate Squ. hyperplasia: Squamous hyperplasia Alve. histiocyt. :alveolar histiocytosis Mononuc. infiltr: Mononuclear infiltrate Vasc. minerlisa.: vascular mineralisation Pigmen. macrop.: Pigment macrophage

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL is deemed to be 1000 mg/kg bw/day for males and females based on no adverse effects when tested up to a maximum recommended concentration for repeat dose toxicity studies. 

Executive summary:

In this study, 3-methylpentane-1,5-diol was administered once via oral gavage for 90 days to Wistar rats. Animals (10/sex/group) were administered test article formulations at concentrations of 0 (water for injection), 100, 300, or 1000 mg/kg bw/day. Body weight and food consumption were measured at regular intervals with neurological function examination performed. Clinical pathology evaluations (haematology, clinical chemistry (including thyroid hormones) and urinalysis) were performed with all surviving animals subjected to complete gross necropsy. All control and high dose group animals were subjected to a full histopathology. Spermatogenesis assessments were made on all male animals at necropsy.

No treatment-related effects were seen on survival, clinical signs, haematological and clinical chemistry parameters, urinalysis or in neurological parameters (FOBs), organ weight, histopathology or spermatogenesis.

Whilst no toxicologically relevant findings attributed to the test article were observed in either sex, a statistically significant lower mean score for response to open field grooming, handling, spontaneous activity and rearing supported in high dose group males were observed. A statistically significant lower mean body temperature was measured in the low and mid dose groups. These changes were considered unrelated to test article administration and deemed serendipitous because: i) the effects were not replicated in the high dose group (temperature); ii) there were no significant differences in any functional observations; iii) the effects were not replicated in females.

Under the conditions of this study, the NOAEL is deemed to be 1000 mg/kg bw/day for males and females based on no adverse effects when tested up to a maximum recommended concentration for repeat dose toxicity studies.