3-methylpentane-1,5-diol

Endpoint summary

Description of key information

Two reliable studies are available. In a 90-day rat oral gavage study, a NOAEL of 1000 mg/kg bw/day was determined in males and females based on no adverse effects when tested up to and including 1000 mg/kg bw/day.

Furthermore, a combined reproductive/developmental toxicity screening study in the rat is available, in which disappearance of lipid droplets in hepatocytes and an increase in glycogen and liver weight were seen in females at 1000 mg/kg bw/day, likely to be related to pregnancy. The NOAEL in this study was set at 300 mg/kg bw/day based on adverse effects in pregnant females.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1996-01-12 to 1997-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted study, carried out by Bozo Research Center Inc. (Japan)

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for testing of Chemicals, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

other: Crj: CD(SD) SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: Approximately for male and female (P) 8 wks; (F1) x wks
- Weight at study initiation: (P) Males: 302-326 g; Females: 206-232 g; (F1) Males: x-x g; Females: x-x g
- Housing: Individually bred in a metal cage (W 190 x D 350 x H 170 mm: Lead Engineering Co.,); except hybridization period from day 17 to nursing day 4; During hybridization period from evening to following morning, one male and one female were accommodated in a metal net cage (W 266 x D 266 x H 200 mm: RIKO Electric Industry Co.,)
- Diet (e.g. ad libitum): solid food (NMF: Oriental Yeast Co.,) ad libitum
- Water (e.g. ad libitum): drinking water (Fujimi Water Association: Automatic water supply) ad libitum. During breeding period, drinking water was supplied by water bottle
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5±23.5°C
- Humidity (%): 50±20%
- Air changes (per hr): ventilation frequency 10 to 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day (7 a.m. to 7 p.m.)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The necessary quantity of the test substance was weighed for each administration, and prepared at necessary temperature by dissolving in water for injection. By the way, the test substance was not purity converted, and indicated as original weight.
The administration dose was determined for 0.5 mL/100g

Individual administration solutions were calculated based on, for males, the weight on the day of measurement, and for females, during mating period weight on each measuring day, and during gestation period weight on gestational day 0, 7, 14 and 21, and during nursing day 0.

For the administration period, the forced administration was performed in accordance with the Guideline (OECD Guideline for testing of Chemicals, Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test) using a metal Magen Sonde, once a day between 9 a.m. to 3 p.m.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before administration start for both sexes and at final week of administration to males, twice by gas chromatography for each concentration used, consequently ratio to the indicated value was respectively within range of 99.0 ~ 105.0 %

Duration of treatment / exposure:

Males: 49 days from 14 days before mating to the day before autopsy
Females: 41 to 45 day from 14 days before mating, mating period (5 days maximum), gestation period and nursing day 4 until the day before autopsy

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
other: nominal conc.

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
other: nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
other: nominal conc.

No. of animals per sex per dose:

12 per sex per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

A 2 weeks preliminary test of oral administration using rats was conducted to determine the consentration range (Test No.: 0 -417, dose: 0, 125, 250, 500 and 1000 mg/kg). For the administration of 1000 mg/kg, there was no apparent effect of the test substance in performance status, weight, amount of food consumption, hematological data and blood chemical laboratory data. Therefore, the high-dose in the test was defined to 1000 mg/kg, then subtracting by a common ratio 3, medium and lower doses were respectively set to 300 and 100 mg/kg.

Positive control:

none

Observations and examinations performed and frequency:

Observation, Measurement and Examination Method and Frequency:

1) Males (P)

(1) Observation of Performance Status
For the performance status, symptom of toxicity and behavioral problems were observed everyday 3 times a day, before and right after and 2 hours after the administration. But, for day-off, observation was twice before and after the administration, on the day of the autopsy once before the autopsy.

(2) Measurement of Body Weight
Weight was measured between 8 a.m. to 12:30 p.m. on administration starting day (before administration on day 1), on administration day 4, 8, 11, 15, 22, 29, 36, 43 and on autopsy day (day 50 after the adminstration).

(3) Measurement of Food consumption
The amount of food consumption was measured between 8 a.m. to 12:30 p.m. of the same day with body weight except on autopsy day, calculated from difference with amount of food consumption of previous day.

(4) Hematological Examination
All animals were abstained from food for overnight (16 hours) at slaughtering autopsy of the following day after the completion of the administration, then, underwent an abdominal surgery by ether anesthesia, then collected blood from abdominal aorta.

a)With the use of the collected blood from abdominal aorta in a blood bottle (SB-41: Toa Medical Electronic Co.,), supplemented with anticoagulant (EDTA-2K), the following measurements were performed:

Coulter 8 -parameter Automated Hematology Analyzer (Nikkaki Co.,) used: RBC count [Electric Resistance Change Detection Method], Hemoglobin [Cyanmethemoglobin Method], Hematocrit [Mean RBC volume (µ^3) X RBC count (10^4/mm^3)/10^3], Mean RBC volume [Electric Resistance Change Detection Method], Mean RBC heoglobin [Hemoglobin (g/dl) X 10/RBC count (10^6/mm^3)], Mean RBC hemoglobin concentration (Hemoglobin (g/dl) X 10^2/Hematocrit (%)], Platelet count and WBC count [Electric Resistance Change Detection Method].
Reticulocyte count [Brecher Method], WBC percentate [May-Giemsa staining at microscopy].

b) Take the collected blood in a test tube with 3.8% sodium citrate, after centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma.

Coagulation Analyzer ACL100 (Instrumentation Laboratory) used: Prothrombin time and Activated partial thromboplastin time [Clot Method], Fibrinogen [Thromboplastin Method].

(5) Blood Chemical Test
The following test was performed using collected blood from abdominal aorta at the same time with blood collection for heamtological examination.

a) Take the collected blood in a test tube, after centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma:

Automatic analyuer [Monarch (Instrumentation Laboratory)] used: AIP [Bessey-Lowry Method], Total cholesterol [CEH-COD-POD Method], Trygliceride [GK-GPO-POD Method], Phospholipid t [PLD-ChOD-POD Method], Total bilirubin [Azobilirubin method], Glucose [Hexokinase-GLDH method], Urea nitrogen [Urease-GLDH method], Creatinine [Jaffe method], Sodium and Potassium and Chlorine [In selective electrode method], Calcium [OCPC method], Phosphor [Molybdate method], Total protein [Bluret method].
Ectrophoresis system [CLINISCAN 2 (Helena Kenkyujyo Co.,) used: Protein fraction [Cellulose acetate menbrane elctrophoresis].
A/G ratio [Calculated from protein fraction ratio].

b) The collected blood from abdominal aorta in a tube containing heparin was centrifuged (3000 rpm, 10 minutes), the following measurements were performed with the obtained plasma.

Automatic analyuer [Monarch (Instrumentation Laboratory)] used: GOT [UV-rate assay], LDH [UV-rate method].
Coulter 8 -parameter Automated Hematology Analyzer (Nikkaki Co.,) used: GPT [UV-rate assay].

(6) Autopsy
After all animals were weighed on the following day of the last administration (50 days after the start of administration), and after collection of blood under ether anesthesia, internal organs and tissues were observed macroscopically by autopsy. Furthermore, brain, heart, lung with bronchial, liver, spleen, hypophysis, thyroid with parathyroid, adrenal, thymus, kidney, testicle and epididymis were collected, internal organs and tissues were fixed in 10% formalin conditioned with phosphate buffer solution (1/15M, pH 7.1 -7.4) except testicle and epididymis which were conditioned in Bouin´s solution. Also, died animal (100 mg/kg dose group: 2003) was treated similarly to the survivals.

(7) Measurement of Organ Weight
For organs, heart, thymus, liver, kidney, spleen, testicle and epididymis were weighed, then, relative organ weight was calculated from final body weight.

(8) Histopathological Examination
For all animals, heat, thymus, liver, kidney, spleen, testicle, epididymis and gross abnormal regio at autopsy were paraffin embedded. For all animals of the control group, of the highest-dose group and of dead animal, a segment was made, then observed at the microscope after eosin staining.


2) Female (P)

(1) Observation of General Appearance
For the general appearance, symptom of toxicity and behavioral problems were observed everyday 3 times a day, before and right after the administration and 2 hours after. But, for day-off, observation was twice before and after the administration, on the day of the autopsy once before the autopsy.

(2) Observation of Estrus Cycle
For the observation of estrus cycle, vaginal smear was collected and observed at the microscope everyday from the start of the administration until the confirmation of copulation. During the administration before mating, the vaginal smear images (picture, statues, statue) were classified by proestrus, estrus, postestrus and anestrus, and then estrus manifestation count and days (estrus cycle) from estrus to the following estrus were checked. Besides, the vaginal smear on the day before the administration was excluded from calculation of estrus cycle. During mating period, the presence of spermatozoa was checked.

(3) Measurement of Body Weight
Weight was measured between 8 a.m. to 12:30 p.m. on administration day (before administration on day 1), on administration day 4, 8, 11 and 15, for during gestatation period on gestational day 0, 7, 14 and 21, and during nursing period on nursing day 0 and 4.

(4) Measurement of Food Conumption
The amount of food consumption was checked between 8 a.m. to 12:30 p.m.. on the same day with weight measured, for during gestation period on gestational day 1, 7, 14 and 21, and for during nursing period on nursing day 1 and 4, calculated from difference with amount of food consumption of previous day.

(5) Observation of Delivery and Nursing
The dams were left for natural delivery to observe the presence of anomaly delivery. The copletion of delivery was checked twice everyday from gestational day 21 to 25, when the delivery has completed before 10 a.m. and the day was indicated as nursing day 0. For one case (No. 1106) in the control group, as delivery was not observed before 10 a.m. on the pregnant day 25 after confirmation of coputation, the autopsy was performed after exsanguination under lethal ether anesthesia to verify formation of pregnancy, because implantation was not observed in utero, it was considered to be sterile.
Dams confirmed for the completion of delivery were left to nurse pups born, and continued observation of nursing everyday until the nursing day 4.

(6) Autopsy
At nursing day 4, autopsy was performed for all dams after exsanguination under lethal ether anesthesia to observe internal organs and tissues macroscopically, also, number of corpus luteum and implantations traces were counted as well. Furthermore, brain, heart, lung with bronchial, liver, spleen, hypophysis, thyroid with parathyroid, adrenal, thymus, kidney, ovary, uterine, vagina and gross abnormal regio were collected and fixed in 10 formalin conditioned with phophate buffer solution (1/15M, pH 7.1 -7.4). The sterile animal (control group: 1106) was treated similarly.

(7) Measurment of Organ Weight
For organs, heart, thymus, liver, kidney, spleen and ovary were weighed, then, relative organ weight was calculated from final body weight.

(8) Histopathological Examination
For all animals, heat, thymus, liver, kidney, spleen and ovary were paraffin embedded. For all animals of the conrol group and of the high-dose group, a segment was prepared, then, observed at the microscope after hematoxylin-eosin staining. In addition, for the case of a liver suspected for the effect of the test substance, segment was prepared for whole group and searched similarly. Furthermore, for 2 examples (1109, 1110) of the control group and 2 examples (4102, 4103) of 1000 mg/kg dose group, the liver was searched by PAS staining.


3) Mating of Parent Animals (P)

After the administration during 14 days, one male and one female as one pair of a same group were lived together all-night. The cohabitation period was maximum 14 days until the confirmation of copulation. Copulation was verfied everyday by checking copulatory plug or presence of spermatozoa in vaginal smear, once confirmed female as acopulated animal, the day was indicated as gestational day 0. Because the male supposed to cross (100 mg/kg dos group: 2003) have died, a female of 100 mg/kg dose group (No. 2103) was bred with another male (No. 2002) of the same group who was verified for copulation.


4) Neonate (F1)

(1) Observation of Neonatal Pups
At nursing day 0, survivals and stillborn children were counted and observed for the presence of body surface anomaly. Survival pups were checked for the sex, and after weighed, and all they were left nursed by dams. The stillborn children were fixed and conserved in 10% formalin conditioned with phosphate buffer solution (1/15M, pH 7.1 -7.4) except those who showed marked deterioration of postmortem change.

(2) Observation and Measurement of Nursing Pups
Nursing pups were observed once a day for their life and death. The weight was measured in nursing day 0 (birth day) and day 4. On the nursing day 4, after all nursing pups were exsanguinated under lethal ether anesthesia, by autopsy the presence of anomaly of internal organs was observed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Significant level was determined for 5 and 1%. For the children, the average of one litter was defined as one unit.

Multiple Test:
The test of distribution uniformity of each group was performed by Barlett´s test. Therefore, when the distribution is uniform, the analysis of variance by One Way Layout was performed, and when there is a significant difference between groups, paired comparison test was performed using the Dunett Test when identical number of samples in each group, and the Scheffé test when different number of samples between groups. When the dispersion was not uniform, the Kruskal-Wallis test was performed, and if significant, for the difference of average of the ranks, the Dunett Test was performed for identical number of samples in each group, and the Scheffé test for different number of samples between groups
Body weight, amount of food consumption, estrus manifestation count, estrus cycle, cohabitation days, gestation period, hematological examination, blood chemical test, organ weight, corpa lutea count, implantation traces, number of children born, implantation rate, sex ratio, delivery rate, birth rate and survivla rate in neonatal pups


Chi-Square Test:
Copulation rate, pregnancy rate and childbirth rate


Calculation of Various Data:
Copulation rate (%) = (No. of copulation verfied animals/No. of mating animals) X 100
Conception rate (%) = (No. of pregnant animals/No. of copulation verfied animals) X 100
Childbirth rate (%) = ( No. of dams with live birth/No. of pregnant dams) X 100
Pregnancy period (day) = Nursing day 0 (delivery verified date) - gestation date 0
Sex ratio = Males/(males + females)
Neonatal survival rate (%) = (No. of survivals on nursing day 4/No. of lives on nursing day 0) X 100
Delivery rate (%) = (Total childbirth pups/No. of implantation traces) X 100
Birth rate (%) = (No. of survivals on nursing day =/Total childbirth pups) X 100
Implantation rate (%) = (No. of implantation traces count/No. of corpus luteum) X 100

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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

Details on results:

I. Repeat Dose Toxicity

1. Effect to Males

1)Performance Status
In 7 cases ( No. 4004, 4006, 4007, 4009, 4010, 4011, 4012) of the 1000 mg/kg dose group, salivation was seen 29 days after the administration. This salivation was expressed right after the administration, and continued until at the completion of administration period. This salivation disappeared 1 hours after the administration.
And, 1 case of the 100 mg/kg dose group (No. 2003) had died on the administration day 17. For this case, there was no change in performance status, weights and amount of food consumption until the day before death, though lung became dark reddish, histological alveolus edema and humor retention with pale red foamy substance was seen, the cause of death was estimated to be an incorrect administration. Also, in other case in the control group (No. 1008), a maxillary tooth crack was observed on administration day 32.

2) Weight
The weight in the test substance group has changed during administration period similarly to the control group, and the significant difference in weight on each mesuring day and in weight gain during the administration period was not seen between the control groups.

3) Food Consumption
The amount of food consumption in the test substance administration showed approximately similar value with the control group through the administration period, and the significant difference in amount of food consumption on each measuring day was not seen between the control groups.

4) Hematological Findings
Though a significantly low value of RBC was seen in the 100 mg/kg group, there was no association with the administered dose. Other examination parameters in the test substance dose group showed approximately the same values with control group, and the significant difference was not seen between the contol group and the test substance dose group.

5) Blood Chemical Test
A significant low value of urea nitrogen was seen in the 300 mg/kg dose group, but there was no association with the administration dose. Also, a significant low value of a alpha 1 -globulin fraction was observed was seen in 1000 mg/kg dose group, a slight increase of albumin ratio and A/G ratio in protein fraction were observed in this dose group. Other parameters in the test substance dose group have shown approximately the same value with the conrol group, significant difference was not seen between the conrol group and the test substance dose group.

6) Autopsy Findings
A diffusion of dark reddish spots in lung was observed in one case (No. 4008) of the 1000 mg/kg test group. In other cases, there was no macroscopic abnormaly.

7) Organ Weights
A significant high value of the relative weight of spleen was observed in the 300 mg/kg dose group, but, there was no association with the administered dose. Other, in the absolute and relative weights of the measured organs and tissues, i.e. thymus, herat, kidney, testis and epididymis, the significant difference was not seen between the control group and the test substance dose group.

8) Histopathological Findings
The change related to the test substance administration was not recognized for the histopathological examination carried out for 1000 mg/kg dose group. But, the minor fat development of hepatic cell in liver lobe peripheral increased extramedullary hematopoiesis in spleen were observed in all cases in the control group and the 1000 mg/kg dose group.
In addition, eosinophilic body was found in 2 cases (1002, 1006) in the control group, and in 3 cases (4001, 4003, 4006) in the 100 mg/kg dose group, furthermore, restricted atrophy of seminiferous tubule in testis and restricted infiltration of inflammatory in lung were observed in one case (No. 4008) in the group of 1000 mg/kg dose group, but as these changes were either minor or slight, they were considered contingent from ther manifestation.


2. Effect to Feamales

1) General Appearance
There were no death during before and during mating period, gestational period and through to nursing period.
Change in general apperance before and during mating period was not observed neither in the control group nor in the test substance dose group.
During gestational period, at the observation right after the administration, salivation was seen coninously until 10 days after the gestation, in 5 cases (No. 4101, 4104, 4107, 4110, 4112) of the 1000 mg/kg dose group. This salivation disappeared similarly to males 1 hour after the administration. No other general apperance was observed.
Also during nursing period, similarly to the gestational period, salivation was seen continously from postpartum to nursing day 3 in 5 cases (No. 4104, 4105, 4107, 4110, 4112) of the 1000 mg/kg dose group. No other performance status was observed.

2) Body Weights
The weight in the test substance group has changed similarly to the control group through mating period, gestation period and to nursing period, and the significant difference in weight on each measuring day and in weight gain during each period was not seen between the control group and the test substance dose group.

3) Food Consumption
The amount of food consumption in the group of the test substance dose group has shown through mating period, gestation period and to nursing period, approximately the similar values to the conrtol group, and the significant difference in amount of food consumption on each measuring day was not seen between the control group an the test substance dose group.

4) Autopsy Findings
An adhesion of adipose tissues on spleen, liver, stomach and on periphery to these organs was seen in one case (No. 4108) in 1000 mg/kg dose group. In other case, there was no macroscopic anomaly.

5) Organ Weight
In the group of 1000 mg/kg dose group, a significant high value of absolute and realtive weight in liver and the rise of absolute weight in liver and the rise of abslolute weight in bilateral kidney and a significant high value of its relative weight were seen. Though a significant difference was not seen in the group, the absolute and relative value of thymus have showed a decline trend. Furthermore, in the absolute and relative weight of the measured organs and tissues, i.e. thymus, heart, spleen and ovary, the significant difference was not seen between control group and the test substance dose group.
For the 300 mg/kg dose group, in the absolute and realtive weight of the measured organs and tissues, the significant difference was not seen between the control group and the test substance dose group.

6) Histopathological Findings
In the 1000 mg/kg dose group, a change in liver was seen, which could be associated to the test substance administration. In other words, the increase of glycogen accumulated in hepatic cells was seen slightly in 8 cases in the control group, in 9 cases in the 100 mg/kg dose group, and in 8 cases in 300 mg/kg dose group, but in all cases of 1000 mg/kg dose group, it was minor or slightly, a significant increase of number of mainifestation was recognized between the control group and the 1000 mg/kg dose group. Also, minor fat development of hepatic cell in liver lobe peripheral was seen in 10 cases in the control group, in 6 cases in the 100 mg /kg dose group and 6 cases ub tge 300 mg/kg dose group, as opposed to no observation in the 1000 mg/kg dose group, a significant increase of number of manifestation was recognized between the control group and the 1000 mg/kg dose group. Therefore, in the 1000 mg/kg dose group, it was considered that fat development in hepatic cells disappears, and amount of glycogen accumulated in hepatic cells increases. Besides, similarly to males, a slight increased extramedullary hematopoiesis in spleen was seen in all cases of the control group and of 1000 mg/kg dose group, and slight capsule fibrosing was seen in one case (No.4108) in the 1000 mg/kg dose group which had also adhesion in spleen at autopsy, although the significant difference in manifestation of these findings was not seen between the control group and the test substance dose group, it was considered contingent due to their manifestation.

II. Reproductive Toxicity

1. Effect to Parent Animals

1) Estrus Cycle
In the test substance dose group, the estrus cycle i.e. estrus manifestation count and days from estrus to following estrus was approximately similar to the control group, and the significant difference was not seen between the control group and the test substance dose group.
For each individual, in one case (No. 4108) in the 1000 mg/kg dose group, an anestrus was observed continously, though no anomaly was seen in estrus cycle for other cases. Besides, for one case (No. 4108) in 1000 mg/kg dose group, which showed anomaly of the estrus cycle, copulation was confirmed at mating day 5, no anomaly was recognized for impregnation capability. Also, as there was no other cases of estrus cycle anomaly in the same group, it was considered as a contingent change independent from the test substance administration.

2) Copulation Rate and Impregnation Rate
In either of the control group and of the test substance dose group, copulation was formed within 5 days after cohabitation, the copulation rate in each group was 100%. There was no significant difference in average days required for formation of copulation between the control group and each test substance group. The formation of copulation was observed in all pairs except in one pair (No. 1006 X 1106) in the control group, and the pregnancy rate in the test substance dose group was 100%.

3) Number of Neonatal Pups Birth Females, Gestation Period, and, Nursing Conditions
In one case (No. 1106) in the control group, delivery was not observed even at pregnancy day 25, as a result of autopsy it was steril. Other cases in the control group and in the test substance administration group, completion of delivery was confirmed by pregnancy day 23, but there was no significant difference in pregnancy period between the control group and the test substance dose group. In addition, anomaly of delivery was not observed, and birth rate was 100% for each group. For the nursing conditions, there was no anomaly in nursing behavior such as nesting, pup approach and lactation in every case in the control group and in the test substance group.

4) Corpus Luteum Count, Implantation Traces, Implantation Rate and Number of Neonatal Pups Births
The number of corpus leuteum, implantation traces and implantation rate showed approximately the same value with the control group, there was no significant difference between the control group and the test substance group. Besides, stillbirth were seen only in 3 dams (No. 2101, 2107, 2109) of the 100 mg/kg dose group, and in 2 dams (No. 3103, 3111), number of children born in the test substance dose group showed approximately the same value with the control group, thus there was no significant difference in delivery rate between the control group and the test substance dose group.

2. Effect to Neonatal Pups

1) Sex Ratio, Body Surface Anomaly, Survivals and Existence Rate
There was not significant difference in sex ratio between the control group and the test substance dose group. For the observation of the body surface of neonatal pups, no significant difference was observed between the control group and the test substance dose group. Number of survivals and birth rate in the test substance group showed approximately the similar value with the control group, no significant difference was observed between the control group and the test substance dose group. Additionally, neonatal pups died during up to 4 days after birth were seen in 2 cases in 2 dams (No. 2106, 2111) of the 100 mg/kg dose group, and in 6 cases in 3 dams (No. 3103, 3108, 3109) of the group, though no significant difference was observed in survival rate on the day 4 after birth between the control group and the test substance grouop.

2) Body Weights
The weights on the birth and on the day 4 after birth in the test substance dose group showed approximately similar value with the control group, no significant difference was observed between the control group and the test substance dose group.

3) Autopsy Findings
At the autopsy on the day 4 after birth, anomaly was not seen either in the control group or in the test substance dose group.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

                                      Females 
Dose level (mg/kg/day)                 0            1000
Body weight, day 4 of lactation

           (g, Mean ± SD)      351 ± 24       347 ± 20
Absolute weight
Liver (g, Mean ± SD)          14.77 ± 1.50   16.90 ± 2.00**
Relative weight
Liver (g%, Mean ± SD)          4.20 ± 0.24    4.87 ± 0.36**
Kidney (g%, Mean ± SD) right0. 0.32 ± 0.02    0.35 ± 0.02*
Kidney (g%, Mean ± SD) left    0.33 ± 0.02    0.35 ± 0.02* 

*(p<0.05); **(p<0.01, Significant difference from control

- Histopathology (incidence and severity): 
  Female: 
  Liver: Lack of fat deposits and increased glycogen granule
at 1,000 mg/kg 

Dose level (mg/kg/day)   degree*   0    100    300   1000
No. of animals                    12     12     12     12
Fatty deposit/hepatocyte   0       2      6      6     12*
 /periportal               1      10      6      6      0
                           2       0      0      0      0
glycogen accumulation      0       4      3      4      0
/hepatocyte                1       8      9      8      4
                           2       0      0      0      8*
*degree: 0 negative, 1 slight, 2 mild

Conclusions:

In the 1,000mg/kg group, salivation, which appeared immediately after dosing and lasted for about 1 hour, was observed in approximately half of the animals, in males from day 29 of dosing and in females from day 10 of gestation. Furthermore, a lack of fat deposits and an increase of glycogen accumulation in hepaocytes were recorded in females of the 1,000 mg/kg group on histopathological examination. Based on clinical sign in both sexes and histopathological findings in female at 1,000 mg/kg, the NOAEL of repeat dose toxicity is considered to be 300 mg/kg/day.

Executive summary:

As part of OECD investigation project of toxicity in regard to existing chemical safety review, a combined repeat dose toxicity and reproduction toxicity test of 3 -Methyl-1,5 -Pentanediol by oral administration in rat was performed. In other words, dose 0 (control group), 100 and 300 and 1000 mg/kg of the substance was orally administered to SD rat (Crj=CD); for males, during 49 days from 17 days before mating start through to mating period until the day before autopsy, for females, 41 to 45 days from 14 days before mating start through to mating period, gestation period after copulation formation and delivery until nursing day 3. Then, the effect of general toxicity in male and female animals by repeat administration was searched as well as the effect to reproductive ability and formation and growth of following generation such as the gonad function, copulation behavior, impregnation and delivery and nursing.

Repeat Dose Toxicity:

1. Effect to Males (P)

In the group of 1000 mg/kg administration, for observation of performance status, approximately in a half of cases, salivation was manifested 29 days after the administration. This salivation was expressed right after the administration, and changed to disappear in 1 hour after the administration. Also, in the blood chemical test, a decrease of alpha 1 -globulin fraction ratio and a slight increase of albumin and A/G ratios in protein fraction were observed. The effect by the administration of the test substance was not observed in weight, amount of food consumption, hematological and in pathological findings. In 300 and 100 mg/kg dose groups, effect caused by the administration of the test substance was not observed in any of performance status, body weight, food consumption, hematological, blood chemical and pathological findings.

2. Effect to Females (P)

In the group of 1000 mg/kg, for the observation of performance status, similarly in males, salivation right after the administration was seen in about half of cases at after gestational day 10. Furthermore, in the pathological findings, an increase of glycogen and liver weight involving disappearance of lipid droplet of hepatic cell was observed. In the group of 300 and 1000 mg/kg administration groups, the effect caused by the administration of the test substance was not observed in any of performance status, body weight, amount of food consumption and pathological findings.

Consequently, under the test conditions, the general toxic No Observed Adverse Effect Level (NOAEL) is estimated at 300 mg/kg/day for both males and females.

Reproduction Toxicity

1. Effect to Reproductive Toxicity (P)

In the group of 1000 mg/kg dose group, the effect of the administration of the test substance was not observed in any of female estrus cycle and estrus frequency, sexual copulation and impregnation rates, female gestation period, delivery and nursing status, furthermore, nor in corpus luteum count, implantation traces and implantation rate, number of neonatal pups born and delivering rate.

2. Effect to Neonatal Pups (F1)

Also in the group of 1000 mg/kg dose group, the effect of the administration of the test substance was not observed in number of survival at nursing 0 day, number of stillborn infants and birth rate and sex ratio, there was no neonate with anomaly in body surface. In addition, the effect of the administration of the test substance was not seen in survival rate and body weight on the day 4 after birth, and anomaly was not noted for autopsy as well.

Consequently, the reproductive toxic No Observed Adverse Effect Level (NOAEL), under the test conditions, is estimated at 1000 mg/kg/day for both parental animals and neonatal pups.

Reference 2

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

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

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

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)

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

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

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.

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

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

Critical effects observed:

no

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

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. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

hepatobiliary

Organ:

liver

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

In the 90-day rat oral gavage study, there were no test article related effects on survival, clinical signs, blood haematological and clinical chemistry parameters, urin analysis, in neurological parameters (FOBs and motor activity), organ weight or 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 as observed in the high dose group in males. A statistically significant lower mean body temperature was also 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.

A NOAEL of 1000 mg/kg bw/day was determined in males and females based on no adverse effects when tested up to a maximum recommended concentration for repeat dose toxicity studies. 

In an OECD combined repeat dose and reproductive/developmental toxicity screening test [TG 422], male and female rats received 3 -methyl-1,5 -pentanediol (CAS: 4457 -71 -0) by gavage at doses of 0, 100, 300 and 1,000 mg/kg/day for 49 days for males and 41 -45 days for females. Salivation was observed in both sexes at 1,000 mg/kg. A lack of fat deposits and an increase of glycogen accumulation in the liver accompanied by increased liver weight were observed in the females of the 1,000 mg/kg dose group only. Based on these slight changes, NOAEL is considered to be 300 mg/kg/day.

No repeated dose toxicity data are available for the inhalation or dermal exposure routes for 3 -methyl-1,5 -pentanediol (CAS: 4457 -71 -0).

 

Specific target organ toxicity (CLP Regulation) – Repeated exposure (STOT RE)

Summary and discussion of repeated dose toxicity findings relevant for classification as STOT RE according to CLP Regulation

The short-term repeated-dose toxicity of 3-methylpentane-1,5-diol has been investigated by the oral route in rats following 90 days of exposure in a modern test guideline compliant study, along with a combined repeated dose and reproduction / developmental screening toxicity study with rats dosed for between 41 and 49 days.

 

Classification with STOT- RE is triggered by the occurrence of significant (and/or severe for Category 1) toxic effects at doses below specified guidance values. For STOT-RE Category 2, the relevant guidance values for oral exposure are 100 mg/kg bw/day (rat 90-day study) and 300 mg/kg bw/day (rat 28-day study). 

 

As described in the rat, no treatment-related effects on any organ were seen in the 90-day toxicity study when dosed orally via gavage up to a maximum dose of 1000 mg/kg bw/day, the maximum recommended dose in accordance with current repeat dose mammalian toxicity test guidelines.

 

In the rat combined reproductive/developmental toxicity screening study increased liver weights with associated glycogen accumulation in hepatocytes was observed in both males and females at 1000 mg/kg bw/day, with NOAEL set at 300 mg/kg bw/day.

 

Therefore, in the rat, the only significant toxic effects of relevance to humans were seen in the liver; however, these occurred at dose levels well in excess of the specified guidance values.

Comparison with criteria of repeated dose toxicity findings relevant for classification as STOT RE 

As described above in the rat no significant toxic effects occurred at any dose.

On this basis, classification of 3-methylpentane-1,5-diol with STOT-RE is not warranted. 

Justification for classification or non-classification

Taking all data into account 3-methylpentane-1,5-diol is not classified for repeated dose toxicity under Regulation 1272/2008/EC.