Name: OECD_SIDS / SUBSTANCE : 4-Methylpyridine / 108-89-4 Tue, 29 Nov 2022,
15:17:51+0900 /Legal entity owner: National Institute of Health Sciences
Printing date: 2022-11-29T15:17:51.897+09:00

iTable of Contents
0/0 ............................................................................................................................. 1
National Institute of Health Science ..................................................................... 2
4-Methylpyridine .................................................................................................. 3
1 General information .................................................................................. 3
1.1 Identification ................................................................................. 3
Identification ............................................................................... 3
Identification ...................................................................... 3
7 Toxicological information ......................................................................... 4
7.5 Repeated dose toxicity ................................................................... 4
7.5.1 Repeated dose toxicity: oral ................................................. 4
Repeated dose toxicity: oral. 001 .......................................... 4
7.6 Genetic toxicity ............................................................................ 15
7.6.1 Genetic toxicity in vitro ...................................................... 15
Genetic toxicity in vitro.001 ................................................ 15
Genetic toxicity in vitro.002 ................................................ 21
7.8 Toxicity to reproduction ............................................................... 25
7.8.1 Toxicity to reproduction ..................................................... 25
Toxicity to reproduction. 001 .............................................. 25
References ................................................................................................................ 36
Reference Substances ...................................................................................... 36
4-methylpyridine ........................................................................................ 36
Test Materials .................................................................................................. 37
4-methylpyridine ........................................................................................ 37
Literatures ....................................................................................................... 38
Combined repeated dose toxicity study with the reproductive/
developmental toxicity screening test of 4-methylpyridine by oral
administration in rats ................................................................................ 38
In Vitro Chromosomal Aberration Test of on 4-methylpyridine Cultured
Chinese Hamster Cells. ............................................................................. 39
Reverse Mutation Test of 4-methylpyridine on Bacteria. ............................... 40
Legal Entities ................................................................................................... 41
National Institute of Health Sciences .......................................................... 41ii

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Dossier submission type
Name
OECD SIDS
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Name (given by user)

Dossier subject
Dossier subject
4-Methylpyridine / 108-89-4
Public nameSubmitting legal entity
National Institute of Health Science
Dossier creation date/time
Tue, 29 Nov 2022, 15:17:51+0900
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LEGAL_ENTITY: National Institute of Health
Science
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General information
Legal entity nameNational Institute of Health Science2

4-Methylpyridine
General information
Identification
Identification
SUBSTANCE: 4-Methylpyridine

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Substance name4-Methylpyridine
Legal entity
National Institute of Health Sciences / Kawasaki / Japan

Identification of substance
Reference substance
4-methylpyridine / 108-89-4
EC number

EC name

CAS number

CAS name

108-89-4
IUPAC nameRole in the supply chain
Manufacturer
false
Importer
false
Only representativefalse
Downstream user
false

3

Toxicological information
Repeated dose toxicity
Repeated dose toxicity: oral
ENDPOINT_STUDY_RECORD: Repeated dose toxicity: oral. 001

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Administrative data
Endpoint
short-term repeated dose toxicity: oral
Type of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study OECD Test Guideline study under GLP condition
Reliability 1
Cross-reference
Reason / purpose for cross-reference
reference to same study 7.8.1 Toxicity to reproduction: Toxicity to reproduction. 001
Related information
OECD / Toxicity to reproduction / Toxicity to reproduction. 001 / 4-Methylpyridine / 108-89-4

Data source
Reference
Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test of /Ministry of Health, Labour and Welfare (MHLW), Japan / study report
Data accessdata published https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF108-89-4d.pdf
4Materials and methods
GLP compliance
yes
Limit test
no

Test material
Test material information
4-methylpyridine
Specific details on test material used for the study- Name of test material (as cited in study report): 4-methylpyridine- Analytical purity: 99.0%- Storage condition of test material: Room temperature, shading, airtightness (filled with argon gas)
- Stability under test conditions: The stability of test material was identified by analysis of the remainder.

Test animals
Speciesratcommon rodent species
Strainother: Crl:CD(SD)Sexmale/female
Details on test animals or test system and environmental conditions
TEST ANIMALS- Source: Charles River Japan, Inc., Hino Breeding Center.
- Age at study initiation: 10 weeks old- Weight at study initiation:
Males (main study groups): 375-421 g, females (main study groups): 185-234 g, females in (matinggroups): 207-254 g- Housing: Animals were individually housed in stainless steel suspension cage (240W × 380D × 200Hmm), from gestation day 18 to lactation day 4, Dams were bred individually or with individual litterma
tes in plastic cages (310W x 360D x 175H mm) and bedding.
- Diet: Solid feed (CRF-1: Oriental Yeast Co., ltd.) was given ad libitum.
- Water: Tap water was given ad libitum.
- Acclimation period: Males (main study groups): 18 days, females (main study groups): 19 days,
females (mating groups): 18 days
ENVIRONMENTAL CONDITIONS- Temperature (°C): 20-26°C (actual temperature: 22.2-24.3°C)- Humidity (%): 40.0-70.0% (actual humidity: 41.1-66.8%)- Air changes (per hr): 12- Photoperiod (hrs dark / hrs light): 12 hr dark/12 hr light (light: 6:00~18:00)

Administration / exposure
Route of administration
oral: gavage
5Vehiclewater for injection
Details on oral exposure- Amount of vehicle (if gavage): 5 mL/kg- Dosing volume: 5 mL/kg
Analytical verification of doses or concentrations
yes
Details on analytical verification of doses or concentrations
Concentrations of the test solutions using administration on day 1 were analyzed with GC. Analyticalconcentrations of the test solutions were all within the range of 99.5-100.5% of the nominal concen
trations and both values were within the acceptable range (concentration: percentage of nominal
concentration, 100±10%).
Duration of treatment / exposure
Males: 28 days including 14 days pre-mating
Females (main study groups): 28 days
Females (mating groups): 42-46 days including 14 days pre-mating, mating and gestation periods andthe days until day 4 of lactation
Frequency of treatment
Once/day, 7 days/week
Doses / concentrations
Dose / conc.
0mg/kg bw/day (actual dose received)

Dose / conc.
5mg/kg bw/day (actual dose received)

Dose / conc.
20

mg/kg bw/day (actual dose received)

Dose / conc.
80

mg/kg bw/day (actual dose received)

No. of animals per sex per dose
- Main study groups:
Control- and high-dose groups: 12 males and 10 females per group (half of both sexes assigned as t
he treatment groups, and the remaining half assigned as the recovery groups)
Low- and middle-dose groups: 12 males and 5 females per group (half of males assigned as thetreatment groups, and the remaining half assigned as the recovery groups)- Mating groups:
12 females per dose
Control animals
yes, concurrent vehicle6

Details on study design- Dose selection rationale: Based on the results of a 14-day preliminary study, the high dose was set to
80 mg/kg bw/day, and the intermediate dose and low dose were set to 20 mg/kg bw/day and 5 mg/
kg bw/day, respectively.
[14-day preliminary study]
A 14-day repeated dose oral toxicity test (Crl:CD(SD) rats, doses: 0, 50, 100 or 200 mg/kg bw/day).
All males and females at 200 mg/kg bw/day died or were moribund, and one male at 100 mg/kg
bw/day was moribund. Decreased body weight and food consumption were observed in males
at 100 mg/kg bw/day and above and females at 200 mg/kg bw/day. Decreased platelet counts
were observed in females at 50 mg/kg bw/day and above. Increased in adrenal glands weight wasobserved in females at 100 mg/kg bw/day.
- Rationale for animal assignment (if not random): Body weight-balanced randomization

Examinations
Observations and examinations performed and frequency
CAGE SIDE OBSERVATIONS: Yes- Time schedule:
Males and females (main study groups): 2 times/day (before administration, 6-197 minutes afteradministration) during the administration period. Once a day during the recovery period.
Females (mating groups): 2 times/day (before administration, 8-160 minutes after administration) dur
ing the administration period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule:
Males and females (main study groups: on day of grouping, on days 7, 14, 21 and 27 of administration
period.
Females (mating groups): on day of grouping, on days 7 and 14 of administration period, on days 1, 8
and 15 of gestation period, on day 4 of lactation period.
BODY WEIGHT: Yes- Time schedule for examinations:
Males and females (main study groups:
Twice a week (On days 1, 4, 8, 11, 15, 18, 22, 25, 28 and 29 of administration period, on days 1, 4, 8, 11, 14 and 15 of recovery period).
Females (mating groups): Twice a week (On days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46, 50
and 53 of administration period, on days 0, 7, 14 and 20 of gestation period, on days 0, 4and 5 of lactation period).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption: Yes- Time schedule for examinations:
Males and females (main study groups):
Twice a week (Males: On days 2, 5, 9 and 12 of administration period, on days 2, 5, 9 and 12 of recov
ery period; Females: On days 2, 5, 9, 12, 16, 19, 23 and 26 of administration period, on days 2, 5, 9 and
12 of recovery period).
Females (mating groups): Twice a week (On days 2, 5, 9 and 12 of administration period, on days 2, 9,
16 and 20 of gestation period, on days 2 of lactation period).
WATER INTAKE- Time schedule for examinations:
Males and females (main study groups):
Twice a week (Males: On days 2, 5, 9 and 12 of administration period, on days 2, 5, 9 and 12 of recovery period; Females: On days 2, 5, 9, 12, 16, 19, 23 and 26 of administration period, on days 2, 5, 9
and 12 of recovery period).
Females (mating groups): Twice a week (On days 2, 5, 9 and 12 of administration period, on days 2, 9,
16 and 20 of gestation period, on days 2 of lactation period).
7

OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes- Time schedule for collection of blood:
Males and females (main study groups): At the end of administration period, or at the end of recovery
period in both sexes- Anaesthetic used for blood collection: Pentobarbital sodium- Animals fasted: Yes- How many animals:
At the end of administration period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0, 5,
20, 80 mg/kg bw/day)
At the end of recovery period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0 and 80
mg/kg bw/day)- Parameters examined: red blood cell count, hemoglobin, hematocrit, mean corpuscular volume,
mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte percentage, platelet count, white blood cell count, differential white blood cell count, prothrombin time, activatedpartial thromboplastin time, fibrinogen.
CLINICAL BIOCHEMISTRY: Yes- Time schedule for collection of blood:
Males and females (main study groups): At the end of administration period, or at the end of recovery
period in both sexes- Animals fasted: Yes- How many animals:
At the end of administration period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0, 5,
20, 80 mg/kg bw/day)
At the end of recovery period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0 and 80
mg/kg bw/day)- Parameters checked: ALP, total cholesterol, triglyceride, total bilirubin, glucose, urea nitrogen, cr
eatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin, A/G ratio,
AST, ALT, γ-GT
BLOOD HORMONE: Yes- Time schedule for collection of serum:
Males and females (main study groups: At the end of administration period in both sexes- Animals fasted: Yes- How many animals:
6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0, 5, 20, 80 mg/kg bw/day)- Parameters checked: Triiodothyronine (T3), Thyroxin (T4), and thyroid stimulating hormone (TSH)
URINALYSIS: Yes- Time schedule for collection of urine:
Males and females (main study groups): Before the end of the administration period (day 23 of ad
ministration period) and before the end of recovery (day 12 of recovery period).
- Metabolism cages used for collection of urine: Yes
A urine collector to collect fresh urine samples under fasting but ad libitum drinking conditions,followed by collection of 24-hour urine samples under ad libitum feeding and drinking conditions.
- How many animals:
At the end of administration period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0, 5,
20, 80 mg/kg bw/day)
At the end of recovery period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0 and 80
mg/kg bw/day)- Parameters checked:
Fresh urine: Color, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, sediment24-urine: Specific gravity, urine volume (24-hour volume)
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations:8Males and females (main study groups): Final week of administration (Manipulative test and meas
urement of grip strength: Day 27 of administration, measurement of motor activity: Day 26 of adminis
tration), Day 10 of recovery period (measurement of motor activity: males of main study groups)- Dose groups that were examined: Autopsy animals after the end of the administration period- Battery of functions tested:
1) Manipulative Test. Pupillary reflex, approaching behavior, response to touch, auditory reflex, painreflex2) Measurement of Grip Strength. Following manipulative test, grip strength of forelimb and hind limb
were measured by CPU gauge (San Diego Instruments Inc.).
3) Measurement of Spontaneous Motor Activity. Spontaneous motor activity (Ambulatory and verticalcounts) was measured by Activity Monitor (MED Associates Inc.).
The measurements were collected at 10-minute intervals from 1 hour to 2 hours after administration.
Since effects were observed in males of the main test group, males of the main test group were mea
sured before the end of the recovery period (10 days of recovery) at 10 minute intervals for 1 hour.
Sacrifice and pathology
GROSS PATHOLOGY: Yes
ORGAN WEIGHT: Yes [main study groups: brain, pituitary, salivary glands, thyroids, adrenal gland, thymus, spleen, heart, liver, kidney, testes, epididymides, ventral prostate, seminal vesicles, ovaries,
uterus; females in mating group: ovary, uterus ]HISTOPATHOLOGY: Yes, [main study groups: heart, lung, trachea, liver, pancreas, sublingual gland,
submandibular gland, esophagus, stomach, duodenum, jejunum, ileum (including Peyer’s patch),cecum, colon, rectum, thymus, spleen, mandibular lymph nodes, mesenteric lymph nodes, kidney,urinary bladder, testis, epididymis, ventral prostate, seminal vesicles (including coagulating gland),ovaries, uterus, vagina, pituitary, adrenal glands, thyroid (including parathyroid), cerebrum, cere
bellum, pons, spinal cord, sciatic nerve, eye ball, Harderian gland, sternum and femur (including bonemarrows), muscle (rectus femoris), mammary gland; females in mating group: ovaries, uterus, and v
agina]
Statistics
For quantitative data, homogeneity of variance was tested using Bartlett method first. If the variance
was homogenous, statistical difference between each treatment group and the control group was
analyzed using Dunnett method. If not homogenous, statistical difference between each treatmentgroup and the control group was tested using Steel method. For comparison of quantitative data be
tween two groups in the recovery test, homogeneity of variance was analyzed by F-test. Then, if homo
genous, student's t-test was applied. If not homogenous, Aspin-Welch's t-test was used.
For histopathological findings, statistical analysis was carried out in combination with Steel-test a
nd Cochran-Armitage trend test. Regarding clinical observation (except for frequency of urination, defecation, rearing and grooming) and sensory reactivity, Steel test was applied.

Results and discussion
Results of examinations
Clinical signs
effects observed, non-treatment-related
Description (incidence and severity)
CLINICAL SIGNS:[At the administration period]:
Transient salivation was observed in males and females at 80 mg/kg bw/day. This was considered tobe due to the irritant properties of the test substance.
[At the recovery period]:
There were no changes related to the test substance in any groups.

9DETAILED CLINICAL OBSERVATIONS:[At the administration period]:
Transient salivation was observed in males and females at 80 mg/kg bw/day. This was considered tobe due to the irritant properties of the test substance.
Mortality
mortality observed, treatment-related
Description (incidence)
At 80 mg/kg bw/day, two pregnant females died on GD 23 and 24.
Body weight and weight changes
effects observed, treatment-related
Description (incidence and severity)[At the administration period]:
Decreased tendency in body weights were observed in late of the administration period (day 18, 22, 25
and 28) in males at 80 mg/kg bw/day. Reduced body weight gain was observed in females (mating group) at 20 mg/kg bw/day during the lactation period.
[At the recovery period]:
There were no changes related to the test substance in any groups.
Food consumption and compound intake (if feeding study)
effects observed, treatment-related
Description (incidence and severity)[At the administration period]:
Reduced food consumption was observed in females (mating group) at 20 mg/kg bw/day during thelactation period.
[At the recovery period]:
There were no changes related to the test substance in any groups.
Food efficiency
not examined
Water consumption and compound intake (if drinking water study)
effects observed, treatment-related
Description (incidence and severity)[At the administration period]:
Increased water consumption was observed in females (mating groups) at 80 mg/kg bw/day on days
16 and 21 of gestation.
[At the recovery period]:
There were no changes related to the test substance in any groups.
Ophthalmological findings
not examined
Haematological findings
no effects observed
Clinical biochemistry findings
effects observed, treatment-related
Description (incidence and severity)
Including blood hormones (T3, T4, TSH)
CLINICAL BIOCHEMISTRY:[At the end of administration period]:10

Decrease in chloride was observed in males at 80 mg/kg bw/day. Decrease in potassium wasobserved in females (main study groups) at 80 mg/kg bw/day.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
BLOOD HORMONES:
There were no changes related to the test substance in any groups at the end of administration and recovery periods.
Urinalysis findings
no effects observed
Behaviour (functional findings)
effects observed, treatment-related
Description (incidence and severity)[At the administration period]:
Spontaneous motor activity: Decreased total ambulatory counts were observed in males at 20 mg/kg
bw/day and above, decreased total vertical counts were observed in males of 80 mg/kg bw/day.
[At the recovery period]:
There were no changes related to the test substance in any groups.
Immunological findings
not examined
Organ weight findings including organ / body weight ratios
effects observed, treatment-related
Description (incidence and severity)[At the end of administration period]:
Increase in the relative liver weight was observed in females (main study groups) at 80 mg/kg bw/day. Increases in absolute and relative uterus weights were observed in females (mating groups) at 80 m
g/kg bw/day.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
Gross pathological findings
effects observed, treatment-related
Description (incidence and severity)[At the end of administration period]:
Stomach: Dark red spots on the mucous membrane of the glandular stomach were observed in fe
males (main study groups) at 80 mg/kg bw/day.
[At the end of recovery period]:
There were no changes related to the test substance in any groups.
Neuropathological findings
not examined
Histopathological findings: non-neoplastic
effects observed, treatment-related
Description (incidence and severity)[At the end of administration period]:
Liver: Centrilobular cellular infiltrations were observed in males at 20 mg/kg bw/day and above.
Stomach: Erosion of glandular stomach was observed in males and females (main study groups) at
80 mg/kg bw/day.
11

[At the end of recovery period]:
There were no changes related to the test substance in any groups.
[Dead animals]:
Lung: Cellular infiltration of inflammatory cells was observed in dead parental females (matinggroups) at 80 mg/kg bw/day.
Liver: Centrilobular necrosis of hepatocyte was observed in dead parental females (mating groups) at
80 mg/kg bw/day.
Thymus: Increased apoptosis in the cortex was observed in dead parental females (mating groups) at
80 mg/kg bw/day.
Spleen: Decreased density of lymphocytes in the periarterial lymph sheath (PALS), lymphoid follicle,
marginal zone, decreased area in the PALS, and lymphoid follicle were observed in dead parental
females (mating groups) at 80 mg/kg bw/day.
Mandibular lymph node: Decreased density of lymphocytes in the lymphoid follicle, increased starry
sky macrophages in paracortex were observed in dead parental females (mating groups) at 80 mg/kg
bw/day.
Mesenteric lymph node: Decreased density of lymphocytes in the lymphoid follicle, increased starry sk
y macrophages in paracortex were observed in dead parental females (mating groups) at 80 mg/kg
bw/day.
Histopathological findings: neoplastic
not examined

Effect levels
Key result
true
Dose descriptor
NOAEL
Effect level5

mg/kg bw/day (actual dose received)

Based ontest mat.
Sex
male
Basis for effect level
histopathology: non-neoplastic
At 20 mg/kg bw/day, centrilobular cellular infiltrations of liver were observed in males.
other: Neurobehavoural examination:
At 20 mg/kg bw/day, decreased total ambulatory counts were observed in males.
Key result
true
Dose descriptor
NOAEL (non-mating females)
Effect level20

mg/kg bw/day (actual dose received)
12Based on
test mat.
Sex
femaleBasis for effect level
clinical biochemistry
At 80 mg/kg bw/day, decrease in potassium was observed in non-mating females (main study groups).
histopathology: non-neoplastic
At 80 mg/kg bw/day, erosion of glandular stomach was observed in non-mating females (mainstudy groups).
organ weights and organ / body weight ratios
At 80 mg/kg bw/day, increase in the relative liver weight was observed in non-mating females(main study groups).
Key result
true
Dose descriptor
NOAEL (maternal toxicity)
Effect level5

mg/kg bw/day (actual dose received)

Based ontest mat.
Sex
femaleBasis for effect level
body weight and weight gain
At 20 mg/kg bw/day, decrease in body weight gain was observed in females (mating group) during
the lactation period.
food consumption and compound intake
At 20 mg/kg bw/day, decrease in food consumption was observed in females (mating group) du
ring the lactation period.

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF108-89-4d.pdf

Applicant's summary and conclusion
Conclusions
The NOAELs for repeated-dose toxicity were determined to be 5 mg/kg bw/day for males and 20 mg/
kg bw/day for females (non-mating groups), and the NOAEL for maternal toxicity was determined tobe 5 mg/kg bw/day.
Executive summary13

In the combined repeated dose and reproductive/developmental screening test (OECD TG422), SD rats
were treated orally with the test substance at the doses of 0, 5, 20 and 80 mg/kg bw/day. Males (12animals/dose: 6 animals were treated as a recovery group) were dosed for 28 days including a 14 day
pre-mating period. Females (12 animals/dose) were dosed for 42-46 days including 14 day pre-mating,mating, and gestation periods and days until day 4 of lactation. In addition, as the main study group of
females, 5 or 10 females/group was dosed for 28 days without mating (5 females at 0 and 80 mg/kg
bw/day were treated as recovery groups).
Two pregnant females died at 80 mg/kg bw/day on GD 23 and 24. Transient salivation was observed inmales and females at 80 mg/kg bw/day. This finding was considered to be caused by irritancy unrelated
to the toxicity of the test substance. Decreased tendency in body weights were observed in late of thedosing period (day 18, 22, 25 and 28) in males at 80 mg/kg bw/day. Reduced body weight gain andfood consumption were observed in dams at 20 mg/kg bw/day during the lactation period. Increased
water consumption was observed in females of mating group at 80 mg/kg bw/day on days 16 and 21 ofgestation. Decreased total ambulatory counts were observed in males at 20 mg/kg bw/day and above,
decreased total vertical counts were observed in males of 80 mg/kg bw/day. The following findings
were observed in examination at the end of administration period. Decreased in chloride was observedin males at 80 mg/kg bw/day. Decrease in potassium was observed in females of main study group at
80 mg/kg bw/day. Increase in the relative liver weight was observed in females of main study groupat 80 mg/kg bw/day. Increases in absolute and relative uterus weights were observed in females ofmating group at 80 mg/kg bw/day. Centrilobular cellular infiltrations of liver were observed in males at
20 mg/kg bw/day and above. Erosion of glandular stomach was observed in males and females of main
study group at 80 mg/kg bw/day. Histopathological examination of dead females of mating groups at
80 mg/kg bw/day revealed the following lesions. Cellular infiltration of inflammatory cells of the lungs,
centrilobular necrosis of hepatocyte, increased apoptosis in the cortex of thymus, decreased density oflymphocytes in the periarterial lymph sheath (PALS), lymphoid follicle, marginal zone, decreased area in
the PALS, and lymphoid follicle of the spleen, decreased density of lymphocytes in the lymphoid follicle,
and increased starry sky macrophages in paracortex of the mandibular lymph node and the mesentericlymph node.
Based on the above results, the NOAELs for the repeated dose toxicity of 4-methylpyridine were
determined to be 5 mg/kg bw/day for males and 20 mg/kg bw/day for females (non-mating groups),
and the NOAEL for maternal toxicity was determined to be 5 mg/kg bw/day.

14

Genetic toxicity
Genetic toxicity in vitro
ENDPOINT_STUDY_RECORD: Genetic toxicity in vitro.001

UUID: fe5dbe02-c084-4557-bed1-c7895036cdad
Dossier UUID:
Author:Date: 2021-03-15T16:33:05.000+09:00
Remarks:

Administrative data
Endpoint
in vitro gene mutation study in bacteria
Type of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study under GLP condition
Reliability 1

Data source
Reference
Reverse Mutation Test of 4-methylpyridine on Bacteria. / Ministry of Health, Labour and Welfare(MHLW), Japan / study report
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
15

Guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
in vitro gene mutation study in bacteria
Deviations
no
Qualifieraccording to guideline
Guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
genetic toxicity in vitro, otherDeviationsno
GLP compliance
yesType of assay
bacterial reverse mutation assay
in vitro gene mutation study in bacteria

Test material
Test material information
4-methylpyridine
Specific details on test material used for the study
Purity 99.0%

MethodSpecies / strain
Species / strain / cell typeS. typhimurium TA 1535, TA 1537, TA 98 and TA 100
bacteria
Species / strain / cell type
E. coli WP2 uvr A
bacteria
Metabolic activation
with and without
Metabolic activation system
S9 mix: SD male rat liver, induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose
-S9 mix:
312.5, 625, 1250, 2500, 5000 μg/plate (TA 1535, TA 1537, TA 98 and TA 100 strains)
312.5, 625, 1250, 2500, 5000 μg/plate (WP2uvrA strain)+S9 mix:
312.5, 625, 1250, 2500, 5000 μg/plate (TA 1535, TA 1537, TA 98 and TA 100 strains)
312.5, 625, 1250, 2500, 5000 μg/plate (WP2uvrA strain)

16

Maximum concentration was established based on the result of the preliminary test at concentration
up to 5000 ug/plate. In this test, the growth inhibition was not observed for all strains with or without
S9 mix.
Vehicle / solvent- Vehicle(s)/solvent(s) used: water for injection
Controls
Untreated negative controlsnoNegative solvent / vehicle controls
yes
True negative controlsnoPositive controls
yes
Positive control substance
9-aminoacridine
9-amimoacridine hydrochloride (9AA): -S9 mix: (TA1537)
sodium azide
NaN3: -S9 mix: (TA1535)
furylfuramide2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2): -S9 mix: (TA100, TA98, WP2 uvrA)
other: 2-aminoanthracene (2AA)+S9 mix: (TA1535, TA100, TA98, TA1537 and WP2 uvrA)
Details on test system and experimental conditions
METHOD OF APPLICATION: Preincubation
DURATION- Preincubation period: 20 min at 37°C
- Exposure duration: ca.48 hrs
NUMBER OF PLATES: 3
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY- Method: other: growth inhibition
Evaluation criteria
A chemical was judged to be mutagenic when the mean number of revertant colonies per plateincreased more than twice that of the negative control and when the dose-related and reproducible increase was observed.
StatisticsnoResults and discussion
Test results
Key result
trueSpecies / strain
S. typhimurium TA 1535
17

bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
trueSpecies / strain
S. typhimurium TA 1537
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
trueSpecies / strain
S. typhimurium TA 98
bacteria
Metabolic activation
with and without
18

Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
trueSpecies / strain
S. typhimurium TA 100
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity
Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validity
valid
Key result
trueSpecies / strain
E. coli WP2 uvr A
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity
19

Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validityvalid

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF108-89-4e.pdf

Applicant's summary and conclusion
Conclusions
Negative with or without metabolic activation
Executive summary
In a bacterial reverse mutation assay using Salmonella typhimurium TA100, TA1535, TA98, and TA 1537,
and Escherichia coli WP2uvrA (OECD TG 471), 4-methylpyridine was negative with or without metabolic
activation.

20

ENDPOINT_STUDY_RECORD: Genetic toxicity in vitro.002

UUID: 0301b1d6-460c-4c68-80e6-be9e94161134
Dossier UUID:
Author:Date: 2021-03-15T16:35:22.000+09:00
Remarks:

Administrative data
Endpoint
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study under GLP condition
Reliability 1

Data source
Reference
In Vitro Chromosomal Aberration Test of on 4-methylpyridine Cultured Chinese Hamster Cells. / Ministryof Health, Labour and Welfare (MHLW), Japan / study report
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)in vitro cytogenicity / chromosomal aberration study in mammalian cells (from 26 September 2014)21

Deviations
no
Qualifieraccording to guideline
Guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
genetic toxicity in vitro, otherDeviationsno
GLP compliance
yesType of assay
in vitro mammalian chromosome aberration testin vitro cytogenicity / chromosome aberration study in mammalian cells

Test material
Test material information
4-methylpyridine
Specific details on test material used for the study
Purity: 99.0%

MethodSpecies / strain
Species / strain / cell type
Chinese hamster lung (CHL/IU)
mammalian cell line
Cytokinesis block (if used)colcemid
Metabolic activation
with and without
Metabolic activation system
S9 mix: SD male rat liver, induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose
Cell growth inhibition study-S9 mix (short-term treatment): 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 ug/mL+S9 mix (short-term treatment): 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 ug/mL-S9 mix (continuous treatment, 24hr): 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 ug/mL
Main study-S9 (short-term treatment): 250, 500, 1000 ug/mL+S9 (short-term treatment): 250, 500, 1000 ug/mL-S9 mix (continuous treatment, 24hr): 250, 500, 1000 ug/mL
Vehicle / solvent- Vehicle(s)/solvent(s) used: water for injection
22

Controls
Untreated negative controlsnoNegative solvent / vehicle controls
yes
True negative controlsnoPositive controls
yes
Positive control substanceN-dimethylnitrosamine+S9
mitomycin C-S9Details on test system and experimental conditions
METHOD OF APPLICATION: Exposure duration: [short-term treatment]:6 hrs + 18 hr, [continuous
treatment]: 24 hrs
SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa stain (2 v/v%) for 15 min.
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 + 100 cells /concentration
DETERMINATION OF CYTOTOXICITY- Method: relative total growth
Evaluation criteria
For the evaluation of the frequencies of structural aberrations and of polyploidy induced, the following
criteria were employed. Appearance incidence of cell with chromosomal aberrations: Negative (-):
less than 5%, Equivocal(±): 5% or more and less than 10%, Positive(+): 10% or more
StatisticsnoResults and discussion
Test results
Key result
trueSpecies / strain
Chinese hamster lung (CHL/IU)
mammalian cell line
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity
23

Vehicle controls validity
valid
Untreated negative controls validity
not examined
True negative controls validity
not examined
Positive controls validityvalid

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF108-89-4f.pdf

Applicant's summary and conclusion
Conclusions
Negative with or without metabolic activation
Executive summary
In an in vitro chromosomal aberration test using CHL/IU cells (OECD TG 473), 4-methylpyridine wasnegative with or without metabolic activation.

24

Toxicity to reproduction
Toxicity to reproduction
ENDPOINT_STUDY_RECORD: Toxicity to reproduction. 001

UUID: 55509078-56a9-4ba4-94d1-ea3ee3767720
Dossier UUID:
Author:Date: 2022-11-29T14:26:06.844+09:00
Remarks:

Administrative data
Endpoint
reproductive toxicity, other A combined repeated dose/reproductive developmental toxicity study
Type of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study OECD Test Guideline study under GLP condition
Reliability 1
Cross-reference
Reason / purpose for cross-reference
reference to same study
Related information
OECD / Repeated dose toxicity: oral / Repeated dose toxicity: oral. 001 / 4-Methylpyridine / 108-89-4

Data source
Reference
Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test of /Ministry of Health, Labour and Welfare (MHLW), Japan / study report
Data accessdata published https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF108-89-4d.pdf

Materials and methods
25Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test)
Deviationsno
GLP compliance
yes
Limit test
no

Test material
Test material information
4-methylpyridine
Specific details on test material used for the study- Name of test material (as cited in study report): 4-methylpyridine- Analytical purity: 99.0%- Storage condition of test material: Room temperature, shading, airtightness (filled with argon gas)
- Stability under test conditions: The stability of test material was identified by analysis of the remainder.

Test animals
Speciesrat
Strainother: Crl:CD(SD)Sexmale/female
Details on test animals or test system and environmental conditions
TEST ANIMALS- Source: Charles River Japan, Inc., Hino Breeding Center.
- Age at study initiation: 10 weeks old- Weight at study initiation:
Males (main study groups): 375-421 g, females (main study groups): 185-234 g, females in (mating
study groups): 207-254 g- Housing: Animals were individually housed in stainless steel suspension cage (240W × 380D × 2
00H mm), from gestation day 18 to lactation day 4, Dams were bred individually or with individual littermates in plastic cages (310W x 360D x 175H mm) and bedding.
- Diet: Solid feed (CRF-1: Oriental Yeast Co., ltd.) was given ad libitum.
- Water: Tap water was given ad libitum.
- Acclimation period: Males (main study groups): 18 days, females (main study groups): 19 days, females (mating study groups): 18 days
ENVIRONMENTAL CONDITIONS- Temperature (°C): 20-26°C (actual temperature: 22.2-24.3°C)- Humidity (%): 40.0-70.0% (actual humidity: 41.1-66.8%)- Air changes (per hr): 12
26

- Photoperiod (hrs dark / hrs light): 12 hr dark/12 hr light (light: 6:00~18:00)

Administration / exposure
Route of administration
oral: gavage
Vehiclewater for injection
Details on exposure- Amount of vehicle (if gavage): 5 mL/kg- Dosing volume: 5 mL/kg
Details on mating procedure- M/F ratio per cage:1/1- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations
yes
Details on analytical verification of doses or concentrations
Concentrations of the test solutions using administration on day 1 were analyzed with GC. Analyticalconcentrations of the test solutions were all within the range of 99.5-100.5% of the nominal concen
trations and both values were within the acceptable range (concentration: percentage of nominal
concentration, 100±10%).
Duration of treatment / exposure
Males: 28 days including 14 days pre-mating
Females (main study groups): 28 days
Females (mating groups): 42-46 days including 14 days pre-mating, mating and gestation periods andthe days until day 4 of lactation
Frequency of treatment
Once/day, 7 days/week
Doses / concentrations
Dose / conc.
0mg/kg bw/day (actual dose received)

Dose / conc.
5mg/kg bw/day (actual dose received)

Dose / conc.
20

mg/kg bw/day (actual dose received)

Dose / conc.
80

mg/kg bw/day (actual dose received)

27

No. of animals per sex per dose
- Main study groups:
Control- and high-dose groups: 12 males and 10 females per group (half of both sexes assigned as t
he treatment groups, and the remaining half assigned as the recovery groups)
Low- and middle-dose groups: 12 males and 5 females per group (half of males assigned as thetreatment groups, and the remaining half assigned as the recovery groups)- Mating groups:
12 females per dose
Control animals
yes, concurrent vehicle
Details on study design- Dose selection rationale: Based on the results of a 14-day preliminary study, the high dose was set to
80 mg/kg bw/day, and the intermediate dose and low dose were set to 20 mg/kg bw/day and 5 mg/
kg bw/day, respectively.
[14-day preliminary study]
A 14-day repeated dose oral toxicity test (Crl:CD(SD) rats, doses: 0, 50, 100 or 200 mg/kg bw/day).
All males and females at 200 mg/kg bw/day died or were moribund, and one male at 100 mg/kg
bw/day was moribund. Decreased body weight and food consumption were observed in males
at 100 mg/kg bw/day and above and females at 200 mg/kg bw/day. Decreased platelet counts
were observed in females at 50 mg/kg bw/day and above. Increased in adrenal glands weight wasobserved in females at 100 mg/kg bw/day.
- Rationale for animal assignment (if not random): Body weight-balanced randomization

Examinations
Parental animals: Observations and examinations
CAGE SIDE OBSERVATIONS: Yes- Time schedule:
Males and females (main study groups): 2 times/day (before administration, 6-197 minutes afteradministration) during the administration period. Once a day during the recovery period.
Females (mating groups): 2 times/day (before administration, 8-160 minutes after administration) dur
ing the administration period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule:
Males and females (main study groups: on day of grouping, on days 7, 14, 21 and 27 of administration
period.
Females (mating groups): on day of grouping, on days 7 and 14 of administration period, on days 1, 8
and 15 of gestation period, on day 4 of lactation period.
BODY WEIGHT: Yes- Time schedule for examinations:
Males and females (main study groups:
Twice a week (On days 1, 4, 8, 11, 15, 18, 22, 25, 28 and 29 of administration period, on days 1, 4, 8, 11, 14 and 15 of recovery period).
Females (mating groups): Twice a week (On days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39, 43, 46, 50
and 53 of administration period, on days 0, 7, 14 and 20 of gestation period, on days 0, 4and 5 of lactation period).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption: Yes- Time schedule for examinations:
Males and females (main study groups):
28

Twice a week (Males: On days 2, 5, 9 and 12 of administration period, on days 2, 5, 9 and 12 of recov
ery period; Females: On days 2, 5, 9, 12, 16, 19, 23 and 26 of administration period, on days 2, 5, 9 and
12 of recovery period).
Females (mating groups): Twice a week (On days 2, 5, 9 and 12 of administration period, on days 2, 9,
16 and 20 of gestation period, on days 2 of lactation period).
WATER INTAKE- Time schedule for examinations:
Males and females (main study groups):
Twice a week (Males: On days 2, 5, 9 and 12 of administration period, on days 2, 5, 9 and 12 of recovery period; Females: On days 2, 5, 9, 12, 16, 19, 23 and 26 of administration period, on days 2, 5, 9
and 12 of recovery period).
Females (mating groups): Twice a week (On days 2, 5, 9 and 12 of administration period, on days 2, 9,
16 and 20 of gestation period, on days 2 of lactation period).
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes- Time schedule for collection of blood:
Males and females (main study groups): At the end of administration period, or at the end of recovery
period in both sexes- Anaesthetic used for blood collection: Pentobarbital sodium- Animals fasted: Yes- How many animals:
At the end of administration period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0, 5,
20, 80 mg/kg bw/day)
At the end of recovery period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0 and 80
mg/kg bw/day)- Parameters examined: red blood cell count, hemoglobin, hematocrit, mean corpuscular volume,
mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte percentage, platelet count, white blood cell count, differential white blood cell count, prothrombin time, activatedpartial thromboplastin time, fibrinogen.
CLINICAL BIOCHEMISTRY: Yes- Time schedule for collection of blood:
Males and females (main study groups): At the end of administration period, or at the end of recovery
period in both sexes- Animals fasted: Yes- How many animals:
At the end of administration period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0, 5,
20, 80 mg/kg bw/day)
At the end of recovery period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0 and 80
mg/kg bw/day)- Parameters checked: ALP, total cholesterol, triglyceride, total bilirubin, glucose, urea nitrogen, cr
eatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin, A/G ratio,
AST, ALT, γ-GT
BLOOD HORMONE: Yes- Time schedule for collection of serum:
Males and females (main study groups: At the end of administration period in both sexes- Animals fasted: Yes- How many animals:
6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0, 5, 20, 80 mg/kg bw/day)- Parameters checked: Triiodothyronine (T3), Thyroxin (T4), and thyroid stimulating hormone (TSH)
URINALYSIS: Yes- Time schedule for collection of urine:
Males and females (main study groups): Before the end of the administration period (day 23 of ad
ministration period) and before the end of recovery (day 12 of recovery period).
- Metabolism cages used for collection of urine: Yes
29

A urine collector to collect fresh urine samples under fasting but ad libitum drinking conditions,followed by collection of 24-hour urine samples under ad libitum feeding and drinking conditions.
- How many animals:
At the end of administration period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0, 5,
20, 80 mg/kg bw/day)
At the end of recovery period: 6 males/dose (0, 5, 20, 80 mg/kg bw/day), 5 females/dose (0 and 80
mg/kg bw/day)- Parameters checked:
Fresh urine: Color, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen, sediment24-urine: Specific gravity, urine volume (24-hour volume)
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations:
Males and females (main study groups): Final week of administration (Manipulative test and meas
urement of grip strength: Day 27 of administration, measurement of motor activity: Day 26 of adminis
tration), Day 10 of recovery period (measurement of motor activity: males of main study groups)- Dose groups that were examined: Autopsy animals after the end of the administration period- Battery of functions tested:
1) Manipulative Test. Pupillary reflex, approaching behavior, response to touch, auditory reflex, painreflex2) Measurement of Grip Strength. Following manipulative test, grip strength of forelimb and hind limb
were measured by CPU gauge (San Diego Instruments Inc.).
3) Measurement of Spontaneous Motor Activity. Spontaneous motor activity (Ambulatory and verticalcounts) was measured by Activity Monitor (MED Associates Inc.).
The measurements were collected at 10-minute intervals from 1 hour to 2 hours after administration.
Since effects were observed in males of the main test group, males of the main test group were mea
sured before the end of the recovery period (10 days of recovery) at 10 minute intervals for 1 hour.
Oestrous cyclicity (parental animals)
Vaginal smears were collected from all females in the mating groups and microscopically examined
every day from the day after the start of administration until the day copulation was confirmed.
Sperm parameters (parental animals)
Parameters examined in all P male parental generations: testis, epididymis and seminal vesicle weight, histopathological examinations for testes, epididymides, seminal vesicle and ventral prostate.
Litter observations
PARAMETERS EXAMINED: The following parameters were examined in F1 offspring: Number and sexof pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, and weight gain.
GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities.
Postmortem examinations (parental animals)
METHOD OF SACRIFICED: All animals were sacrificed by exsanguination under pentobarbital sodium
anesthesia.
SACRIFICE: Males and females (main study groups): On next day after the last administration,
Maternal animals: on Day 5 of lactation, and males and females recovery group: on Day 14 of recovery.
GROSS PATHOLOGY: Yes
ORGAN WEIGHT: Yes [main study groups: brain, pituitary, salivary glands, thyroids, adrenal gland,
thymus, spleen, heart, liver, kidney, testes, epididymides, ventral prostate, seminal vesicles, ovaries,
uterus; females in mating groups: ovary, uterus]
HISTOPATHOLOGY: Yes, [main study groups: heart, lung, trachea, liver, pancreas, sublingual gland,
submandibular gland, esophagus, stomach, duodenum, jejunum, ileum (including Peyer’s patch), cecu
m, colon, rectum, thymus, spleen, mandibular lymph nodes, mesenteric lymph nodes, kidney, urinary
bladder, testis, epididymis, ventral prostate, seminal vesicles (including coagulating gland), ovaries,
uterus, vagina, pituitary, adrenal glands, thyroid (including parathyroid), cerebrum, cerebellum, pons,
30

spinal cord, sciatic nerve, eye ball, Harderian gland, sternum and femur (including bone marrows),
muscle (rectus femoris), mammary gland; females in mating groups: ovaries, uterus, vagina ]Postmortem examinations (offspring)
SACRIFICE- The F1 offsprings were euthanized on PND4 by exsanguination under 20%Isoflurane anesthesia.
GROSS NECROPSY: Yes- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, andabdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGTHS- Not examined.
Statistics
For quantitative data, homogeneity of variance was tested using Bartlett method first. If the variance
was homogenous, statistical difference between each treatment group and the control group was
analyzed using Dunnett method. If not homogenous, statistical difference between each treatmentgroup and the control group was tested using Steel method. For comparison of quantitative data be
tween two groups in the recovery test, homogeneity of variance was analyzed by F-test. Then, if homo
genous, student's t-test was applied. If not homogenous, Aspin-Welch's t-test was used.
For histopathological findings, statistical analysis was carried out in combination with Steel-test a
nd Cochran-Armitage trend test. Regarding clinical observation (except for frequency of urination, defecation, rearing and grooming) and sensory reactivity, Steel test was applied. Regarding implantat
ion index, delivery index, birth index, live birth index, viability index, sex ratio and external abnormalities, Steel test was performed between administration groups and control groups. Regarding copulation,
fertility index, and gestation index, Fisher's test was applied.
Reproductive indices
Each parameter was determined by the following equations:
Copulation index (%) = (No. of pairs with successful copulation / No. of pairs) × 100
Fertility index (%) = (No. of pregnant females / No. of pairs with successful copulation) × 100
Gestation index (%) = (No. of dams having live pups / No. of pregnant dams) × 100
Length of gestation (days)
Implantation index (%) = (No. of implantation scars / No. of corpora lutea) × 100
Delivery index (%) = (No. of pups born / No. of implantation scars) × 100
Birth index (%) = (No. of live pups born / No. of implantation scars) × 100
Live birth index (%) = (No. of live pups born / No. of pups born) × 100
Sex ratio on Day 4 of lactation = No. of male pups / No. of female pups
External abnormalities (%) = (No. of pups with external abnormalities / No. of live pups) × 100
Offspring viability indices
Viability index (%) = (No. of live pups on Day 4 of lactation/ No. of live pups born) × 100

Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
Clinical signs
effects observed, non-treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
31

Mortality
mortality observed, treatment-related
Description (incidence)See 7.5.1 Repeated dose toxicity. 001
Body weight and weight changes
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
Food consumption and compound intake (if feeding study)
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
Food efficiency
not examined
Water consumption and compound intake (if drinking water study)
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
Ophthalmological findings
not examined
Haematological findings
no effects observed
Clinical biochemistry findings
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
Urinalysis findings
no effects observed
Behaviour (functional findings)
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
Immunological findings
not examined
Organ weight findings including organ / body weight ratios
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
Gross pathological findings
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
32

Neuropathological findings
not examined
Histopathological findings: non-neoplastic
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1 Repeated dose toxicity. 001
Histopathological findings: neoplastic
not examined

Reproductive function / performance (P0)
Reproductive function: oestrous cycle
effects observed, treatment-related
Reproductive function: sperm measures
no effects observed
Reproductive performance
effects observed, treatment-related

Details on results (P0)General toxicity: See 7.5.1 Repeated dose toxicity. 001
Reproductive function / performance: Decrease in number of estrous cases before pairing wasobserved in females at 80 mg/kg bw/day. Prolonged gestation length, decreased tendency in thegestation index, prolonged delivery period, cannibalism and faulty nest-building were observed at 80
mg/kg bw/day.
There were no significant differences in the fertility index, number of corpora lutea, implantation scars
and implantation index between the control group and any treatment groups.

Effect levels (P0)
Key result
false
Dose descriptor
NOAEL
Effect level80

mg/kg bw/day (actual dose received)

Based ontest mat.
Sex
male
Basis for effect level
other: No effects observed on reproduction.
Key result
false
33

Dose descriptor
NOAEL
Effect level20

mg/kg bw/day (actual dose received)

Based ontest mat.
Sex
femaleBasis for effect level
reproductive function (oestrous cycle)
Decrease in number of estrous cases before pairing was observed in females at 80 mg/kg bw/day.
reproductive performance
Prolonged gestation length, decreased tendency in the gestation index, prolonged delivery period,
cannibalism and faulty nest-building were observed at 80 mg/kg bw/day.

Results: F1 generation
General toxicity (F1)Clinical signs
no effects observed
Mortality / viability
mortality observed, treatment-related
Body weight and weight changes
no effects observed
Gross pathological findings
no effects observed

Details on results (F1)
At 20 mg/kg bw/day and above, decreases in numbers of live pups and viability index on lactation day
4 were observed. At 80 mg/kg bw/day, an increase in the number of stillbirths, decreases in the num
ber of live pups, the birth index and the live birth index were observed on lactation day 0.

Effect levels (F1)
Key result
true
Dose descriptor
NOAEL
Generation
F1
Effect level5

mg/kg bw/day (actual dose received)34Based on
test mat.
Sexmale/female
Basis for effect levelviability
At 20 mg/kg bw/day, decrease tendencies in the number of live pups and the viability index onlactation day 4 were observed.

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF108-89-4d.pdf

Applicant's summary and conclusion
Conclusions
In the combined repeated oral dose toxicity study with the reproduction/developmental toxicity screening test (OECD TG 422) described above, there were no reproductive effects in the male parent at
80 mg/kg bw/day, decreased number of estrus cases before pairing, prolonged gestation period,
decreased tendency in the gestation index, prolonged delivery period, cannibalism and faulty nestbuilding, increased absolute and relative weights of uterus were observed in the female parent at 80
mg/kg bw/day and decreased number of live pups and viability on day 4 of nursing in the offspring at
20 mg/kg bw/day and above.
The NOAEL for reproductive/developmental toxicity of 4-methylpyridine was determined to be 5 mg/
kg bw/day based on the reduced number of live pups and survival index observed on day 4 of lactation at 20 mg/kg bw/day, a dose at which reduced body weight gain and food consumption wereobserved in dams.

35

References
Reference Substances
REFERENCE_SUBSTANCE: 4-methylpyridine
UUID: 6be2f443-a8d8-4751-a63f-96736060d0a1
Dossier UUID:
Author:Date: 2020-12-29T17:25:24.000+09:00
Remarks:
Reference substance name
4-methylpyridine

Inventory
CAS number
108-89-4

Molecular and structural information
Molecular formula
C6H7N
Molecular weight
93.13

36

Test Materials
TEST_MATERIAL_INFORMATION: 4-methylpyridine
UUID: 7f8ae6a5-1b8c-45fb-a373-c4eab3e7f09e
Dossier UUID:
Author:Date: 2020-12-29T17:26:15.000+09:00
Remarks:Name
4-methylpyridine

Composition
Composition
Reference substance
4-methylpyridine / 108-89-4
EC number

EC name

CAS number

CAS name

108-89-4
IUPAC nameConcentration99% (w/w)37

Literatures
LITERATURE: Combined repeated dose toxicity study withthe reproductive/developmental toxicity screening test of
4-methylpyridine by oral administration in rats
UUID: 01eaeffa-6516-4bb9-965f-7c1e7a7a32d9
Dossier UUID:
Author:Date: 2020-12-29T17:20:25.000+09:00
Remarks:

General information
Reference Typestudy report
Title
Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test of
4-methylpyridine by oral administration in rats
Author
Ministry of Health, Labour and Welfare (MHLW), Japan
Year
2013
Bibliographic source
available in the web of Japan Existing Chemical Data Base (JECDB) https://dra4.nihs.go.jp/
mhlw_data/home/pdf/PDF108-89-4d.pdf
Testing facility
Nihon Bioresearch Inc.
Report number
10053038

LITERATURE: In Vitro Chromosomal Aberration Test of on
4-methylpyridine Cultured Chinese Hamster Cells.
UUID: 8cc333b3-8235-4004-9818-ddc9ad0f8046
Dossier UUID:
Author:Date: 2021-03-10T11:14:30.000+09:00
Remarks:

General information
Reference Typestudy report
Title
In Vitro Chromosomal Aberration Test of on 4-methylpyridine Cultured Chinese Hamster Cells.
Author
Ministry of Health, Labour and Welfare (MHLW), Japan
Year
2011
Bibliographic source
Japan Existing Chemical Data Base (JECDB)
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF108-89-4f.pdf
Testing facility
Nihon Bioresearch Inc.
Report number
97093039

LITERATURE: Reverse Mutation Test of 4-methylpyridine
on Bacteria.
UUID: a47009b2-5e3c-49de-816c-e6b376899e3f
Dossier UUID:
Author:Date: 2021-03-10T09:57:50.000+09:00
Remarks:

General information
Reference Typestudy report
Title
Reverse Mutation Test of 4-methylpyridine on Bacteria.
Author
Ministry of Health, Labour and Welfare (MHLW), Japan
Year
2011
Bibliographic source
Japan Existing Chemical Data Base (JECDB)
https://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF108-89-4e.pdf
Testing facility
Nihon Bioresearch Inc.
Report number
90133040Legal Entities
LEGAL_ENTITY: National Institute of Health Sciences
UUID: IUC4-b036ff75-0f3c-323b-b200-ed5f46cf5101
Dossier UUID:
Author:Date: 2022-11-07T15:49:29.000+09:00
Remarks:

General information
Legal entity nameNational Institute of Health Sciences
Remarks
Disclaimer: The contents in this document were created based on the MHLW (Ministry of Health,Labour and Welfare) peer reviewed study reports (in Japanese) in JECDB (Japan Existing Chemical
Database) at http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp. Authorship is in the Divisionof Risk Assessment, the National Institute of Health Sciences, and the contents do not reflect any official MHLW opinions or any other regulatory policies.

Address
Address 1
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Postal code
210-9501
Town
KawasakiRegion / State
Kanagawa
CountryJapan
JP

Identifiers
Other IT system identifiers
IT system
LEO
ID
10767
IT systemIUCLID441

ID
16558402024DIV750

42