Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No adverse effects on fertily or organs with reproductive function was observed in rats up to the limit doses in screening studies (OECD 422) and in subchronic oral toxicity studies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
July 2017 - Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility. Analyses demonstrated the stability of the test substance preparations over a period of 10 days at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance solutions in drinking water were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a calibrated
beaker depending on the dose group, topped up with drinking water and intensely mixed with a magnetic stirrer until it was completely dissolved.

FORM AS APPLIED IN THE TEST (if different from that of starting material): mixed with drinking water
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks (male animals); 10 weeks (female animals)
- Weight at study initiation: males: 390 g (mean); females: 215 g (mean)
- Housing: up to 5 animals per sex and cage during pretreatment; individually during the study period
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms. The food used in the study was assayed for chemical and microbiological contaminants. On the basis of the analytical findings the drinking water was found to be suitable. With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

From:27 June 2017 To: 17 Aug 2017 (males); 14 Sept 2017 (females)
Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
ultra-pure
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 1 sample were taken from the low, mid and high concentration for a concentration control analysis. The samples of the gestation were analyzed only if any imprecision occurs during the analysis of the samples from the beginning and lactation of the study.

The samples collected from the beginning of the administration period and during the lactation period were analyzed via capillary gas chromatography with internal standard quantification. The analytical investigations of the test substance preparations were carried out in compliance with the Principles of Good Laboratory Practice.
The test substance concentrations in the drinking water were found to be in the range of 90-110 % of the nominal concentration.
Duration of treatment / exposure:
males: 29 days; females: 58/64 days
The duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 2 days postmating in males as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.
Frequency of treatment:
continuously via drinking water
Dose / conc.:
12 500 ppm
Remarks:
corresponds to a mean daily intake of approx. 785 mg/kg bw/d in males and 1273 mg/kg bw/d in females;
During lactation the concentration was reduced to 50% (6250 ppm) in females.
Dose / conc.:
3 750 ppm
Remarks:
corresponds to a mean daily intake of approx. 229 mg/kg bw/d in males and 359 mg/kg bw/d in females;
During lactation the concentration was reduced to 50% (1875 ppm) in females.
Dose / conc.:
1 250 ppm
Remarks:
corresponds to a mean daily intake of approx. 71 mg/kg bw/d in males and 116 mg/kg bw/d in females;
During lactation the concentration was reduced to 50% (625 ppm) in females.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: A test study was performed in male and female Wistar rats. The test substances (2-Methylbutanol, 3-Methylbutanol, 1-Hexanol and 2-Hexanol) were orally (gavage) applied at dose levels of 0 mg/kg bw/day (corn oil; test group 0), 1000 mg/kg bw/day (2-Methylbutanol; test group 1), 1000 mg/kg bw/day (3-Methylbutanol; test group 2), 500 mg/kg bw/day (1-Hexanol; test group 3) and 500 mg/kg bw/day (2-Hexanol; test group 4) over a period of 14 days to 3 female Wistar rats per test group. After treatment with 3-Methylbutanol (1000 mg/kg bw/d) one animal was found dead on day 5. No mortality occurred in the other test groups. In all treatment groups treatment-related changes of the body weights were observed.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week at the same time of the day (in the morning) until sacrifice, with the following exceptions:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 4, 7, 10, 14, 17 and 20.
• Females with litter were weighed on the day after parturition (PND 1), 4, 7, 10 and 13.
• Body weight was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: twice a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 4, 4 - 7, 7 - 10, 10 - 14, 14 - 17 and 17 - 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
• Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice a week with the following exceptions:
• During pregnancy, water consumption of the females with evidence of sperm was determined for GD 0 - 1, 3 - 4, 6 - 7, 9 - 10, 13 - 14, 16 - 17 and 19 - 20.
• During lactation, water consumption of the females, which gave birth to a litter was determined for PND 1 - 3, 3 - 4, 6 - 7, 9 - 10 and 12 - 13.
• Water consumption was not determined for females without positive evidence of sperm during mating and gestation and for females without litter during lactation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 28 (males) and 55 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/
stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response),
coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

Motor activity assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all surviving males at termination
- Parameters checked in table 3 were examined.
Oestrous cyclicity (parental animals):
In all parental females in the premating phase, estrous cycle normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating. The evaluation continued throughout the pairing period until the female showed evidence of copulation. Additionally, on the day of scheduled sacrifice, the estrous cycle stage was also determined in all female F0 rats.
Sperm parameters (parental animals):
Parameters examined in parental males:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were sacrificed under isoflurane anesthesia by decapitation, blood was sampled for determination of thyroid hormone concentrations and pups were examined externally, eviscerated and their organs were assessed macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, postnatal mortality, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups, blood thyroid hormone concentrations, gross necropsy

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, on study day 29
- Maternal animals: All surviving animals, on study day 58/56

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic (if required) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing.
Statistics:
see table 5
Reproductive indices:
Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- mating, fertility and gestation indices, live birth index, postimplantation loss were calculated for F1 litters (for formulas see "Any other information on materials and methods")
Offspring viability indices:
- viability index, survival index (for formulas see "Any other information on materials and methods")
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption of F0 parental female rats of test group 1 (1250 ppm) was comparable to the concurrent control values throughout the entire study. During gestation, but not premating lactation, an overall slightly higher water consumption was noted for the the mid- (3750 ppm) and high-dose F0 females (12500 ppm, statistically significant during GD 6 - 7 and 16 - 17). This phenomenon may be related to an increased thirst of the females due to the taste/texture of high-concentrated test item solutions. As there were no corroborative signs of toxicity, no adversity is assumed for this finding.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test groups 1, 2 and 3 (1250, 3750 and 12500 ppm) platelet counts were significantly higher compared to controls, which was due to low values in the controls. Additionally, in females of test group 3 (12500 ppm) hemoglobin values were significantly increased. All mentioned values were within historical control ranges (males, platelets 594-776 Giga/L; females, hemoglobin 8.4-9.2 mmol/L). Therefore, these changes were regarded as incidental and not treatment-related. In males of test group 1 (1250 ppm), mean corpuscular hemoglobin (MCH) content and relative neutrophil counts were significantly lower whereas relative lymphocyte counts were significantly higher compared to controls. However, the values were not dose-dependently changed and therefore the changes regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test group 1 (1250 ppm) alanine aminotransferase (ALT) activities were significantly increased, but the values were not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test group 3, the mean absolute brain weight was slightly (-4%), but significantly (p<= 0.05) decreased. Because the mean relative weight was not significantly changed and the mean absolute brain weight (2.066 g) was in the range of historical control data (2.066 g – 2.158 g), a treatment-related effect was considered unlikely.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the cauda epididymidis no treatment-related effects were observed. In males of test group 3 (12500 ppm) sperm head counts in the testis were significantly lower compared to controls. However, the mean was within the historical control range (sperm head counts per gram testis tissue 87-128 Mio/g). Neither any other spermanalysis parameter in these individuals was changed nor any histopathological finding in the testis of these rats was observed. Therefore, the lower sperm head counts in males of test group 3 compared to controls was regarded as incidental and not treatment-related.
The stages of spermatogenesis in the testes of males of the high dose were comparable to those of the controls.
Reproductive performance:
no effects observed
Dose descriptor:
NOAEL
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Remarks on result:
other:
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
None of the individual cases was considered as treatment-related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study. One male runt was seen in test group 3 and one female runt was seen in test group 1.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as diaphragmatic hernia and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Remarks on result:
other: = about 1273 mg/kg bw/d for dams (offspring exposure in-utero and via lactation)
Critical effects observed:
no
Reproductive effects observed:
no

Table 6: Summary Delivery Report

Sex:Female

 

 

 

Test Group 0/ F

0 ppm

 

Test Group 1/ F

1250 ppm

Test Group 2/ F

3750 ppm

Test Group 3/ F

12500 ppm

No. of females at start

N

10

 

10

10

10

No. of females mated

N

10

f-

10

10

10

 

%

100.0

 

100.0

100.0

100.0

Pregnant

N

9

f-

9

10

8

 

%

90.0

 

90.0

100.0

80.0

Without delivery

N

1

 

1

0

2

- Pregnant

N

0

 

0

0

0

- Not pregnant

N

1

 

1

0

2

-- Delivering

N

9

f-

9

10

8

 

%

100.0

 

100.0

100.0

100.0

-- With liveborn pups

N

9

f-

9

10

8

Gestation Index

%

100.0

 

100.0

100.0

100.0

Gestation days

Mean

22.0

n

22.3

22.1

22.1

 

S.d.

0.5

 

0.5

0.3

0.4

 

N

9

 

9

10

8

-- With stillborn pups

N

1

f+

0

2

0

 

%

11

 

0

20

0

-- With all pups stillborn

N

0

f+

0

0

0

 

%

0.0

 

0.0

0.0

0.0

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; n=DUNNETT

Table 7: Summary Litter Report

Test Group 0/ F

0 ppm

 

Test Group 1/ F

1250 ppm

Test Group 2/ F

3750 ppm

Test Group 3/F

12500ppm

Total Number of Pregnant Females

N

9

 

9

10

8

Total number of litters

N

9

 

9

10

8

With liveborn pups

N

9

f-

9

10

8

 

%

100.0

 

100.0

100.0

100.0

With stillborn pups

N

1

f+

0

2

0

 

%

11

 

0

20

0

With all pups stillborn

N

0

f+

0

0

0

 

%

0.0

 

0.0

0.0

0.0

Implantation Sites

N

102

 

104

113

99

 

Mean

11.3

x-

11.6

11.3

12.4

 

S.d.

1.2

 

2.4

2.8

1.9

 

N

9

 

9

10

9

Pups delivered

N

100

 

103

111

97

 

Mean

11.1

x-

11.4

11.1

12.1

 

S.d.

1.2

 

2.5

2.8

2.1

 

N

9

 

9

10

9

Postimplantation Loss

Mean%

1.9

x+

2.0

1.6

2.3

 

S.d.

3.7

 

3.0

3 .4

4.4

 

N

9

 

9

10

9

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

Table 8: Summary Litter Report - Dead Pups

Test

Group 0/ F

0 ppm

Test

Group

1250

1/ F

ppm

Test

Group 2/ F

3750 ppm

Test

Group

12500

3/ F

ppm

Litters with liveborn pups

N

9

9

10

8

Pups delivered

N

100

103

111

97

stillborn / Dead

N

1

0

2

0

 

%

1

0

1.8

0

Alive / Alive

N

99

103

109

97

 

%

99

100

98.2

100

cannibalized / Dead

N

0

0

1

0

 

%

0.0

0

0.9

0.0

sacrificed scheduled / Dead

N

72

72

79

64

 

%

72

68.9

71.2

66

culled / Dead

N

27

32

29

33

 

%

27

32.7

26.1

34

Litters not surviving Day 13

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

Table 9: Summary Litter Report - Pups Died

Test

Group 0/ F

0 ppm

 

Test

Group 1/ F

1250 ppm

Test

Group 2/ F

3750 ppm

Test

Group 3/F

12500ppm

Litters with liveborn pups

N

9

 

9

10

8

Pups delivered

N

100

 

103

111

97

Days 0 To 0

N

0

 

0

0

0

 

%

0

 

0

0

0

Days 1 To 4

N

0

 

0

1

0

 

%

0

 

0

0.9

0

Days 5 To 7

N

0

 

0

0

0

 

%

0

 

0

0

0

Days 8 To 13

N

0

 

0

0

0

 

%

0

 

0

0

0

Pups surviving days 0 To 4

N

99

 

103

108

97

Viability Index

Mean%

100.0

x-

100

99.1

100.0

 

S.d.

0.0

 

0.0

2.9

0.0

 

N

9

 

9

10

8

Pups surviving days 4 To 13

N

72

 

71

79

64

Survival Index

Mean%

100.0

NA

100.0

100.0

100.0

 

S.d.

0.0

 

0.0

0.0

0.0

 

N

9

 

9

10

8

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX; NA=No Test Applicable

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity
in the offspring was 12500 ppm for male (about 785 mg/kg bw/d) and female (about 1273 mg/kg bw/d) Wistar rats, the highest concentration tested.
Executive summary:

In a GLP-compliant OECD 422 study, the test item was administered daily as addition to the drinking water in different concentrations to groups of 10 male and 10 female Wistar rats to screen for potential systemic, reproductive and developmental toxicity. After a two-week premating period, these parental animals were paired and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or PND 13. In males the overall mean dose of the test substance throughout the study was approx. 71 mg/kg bw/d in the 1250 ppm group, approx. 229 mg/kg bw/d in the 3750 ppm group and approx. 785 mg/kg bw/d in the 12500 ppm group; in females it was approx. 116 mg/kg bw/d in the 1250 ppm group, approx. 359 mg/kg bw/d in the 3750 ppm group and approx. 1273 mg/kg bw/d in the 12500 ppm group.

Analyses confirmed the overall accuracy of the prepared concentrations in the drinking water. The stability of these preparations was also demonstrated over a period of 10 days under ambient conditions.

In the clinical examinations of the F0 parental animals no clinical symptoms were caused by the test compound up to the high-concentration of 12500 ppm. In the in-depth investigations including the detailed clinical observation, the functional observational battery and the measurement of motor activity no treatment-related differences to control were observed at any concentration. Water consumption, food consumption and body weights / body weight gain did not show important test substance-related changes.

Concerning clinical pathology (including thyroid hormone measurement) no treatment-related, adverse effects were observed up to a concentration of the compound of 12500 ppm in the drinking water.

Regarding pathology, there were no treatment-related organ weight changes, gross lesions, and histopathological findings in male and female Wistar rats. Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups (1250, 3750, and 12500 ppm) during the entire study. Most F0 parental animals across all test groups proved to be fertile and those individuals failing to generate offspring did not show any specific gross or histopathological findings. Mating behavior, conception, implantation and parturition were not influenced. Pups showed normal development up to scheduled sacrifice. Neither determination of anogenital distance/index not the count of nipple/areola anlagen revealed any treatment-related changes up to and including a concentration of the test item of 12500 ppm in the drinking water.

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 Nov 2007 - 10 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted in compliance with GLP regulations
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley strain SPF rats [Crl:CD(SD)]
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 10 weeks of age
- Weight at study initiation: Males: 338 - 395 g; Females: 217 - 279 g
- Fasting period before study: no
- Housing: individually and subsequently in male-female pairs during the mating period
- Diet (ad libitum): NMF pelleted diet
- Water (ad libitum): tap water (Gotemba City)
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 39 - 73
- Air changes (per hr): 10 to 15 times
- Photoperiod (hrs dark / hrs light): 12-hour lighting per day
Route of administration:
oral: gavage
Vehicle:
other: 1 w/v% CMC solution containing 1% Tween 80 in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A requisite amount of the test article was weighed, suspended in 1 w/v% CMC solution containing 1% Tween 80, transferred to a measuring cylinder, and the vehicle added to make the specified volume to prepare the test suspensions at specified concentrations. Preparation was done at least once in 7 days.

VEHICLE
- Preparation:
A requisite amount of CMC was weighed, dissolved in water for injection and then Tween 80 of the amount equivalent to 1 v/v% of the final volume was dissolved. The mixture was then transferred to a measuring cylinder and water for injection was added to prepare 1 w/v% CMC solution containing 1% Tween 80.

DOSE VOLUME: 10 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: co-housed overnight
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability:
In the stability test of the test suspensions, it was verified that the 100 mg/mL suspension (vehicle: 1 w/v% CMC solution containing 1% Tween 80) was stable at 25°C for 14 days after preparation. The 0.1 mg/mL suspension was verified to be stable at 25°C for 7 days but showed a slight decrease in the concentration after storage for 14 days. Based on these results, the frequency of preparation and storage conditions were selected.

Verification of Concentration and Homogeneity:
Each concentration of test suspensions used for males in week 1 and the final week of administration was analyzed by a GC method (detector: FID) at Gotemba Laboratory, Bozo Research Center Inc. by the method described in “Validation of the analytical method for determination of 3-methylbutan-1-ol in test suspensions by GC: Study No. C-A385”). Results showed that the concentration was 92.7 to 98.8% of the nominal concentration, c.v. value was not more than 3.0%, and both values were within the acceptable range (concentration: percentage of the nominal concentration, 100 ± 10%, c.v.: 10% or below).

Duration of treatment / exposure:
Males: 42 days (14 days before mating, 14 days during the mating period and 14 days after the end of the mating period)
Females: 41 - 53 days (14 days before mating, throughout the mating and gestation periods up to day 4 of lactation)
Satellite (recovery) groups: 42 days
Frequency of treatment:
once a day
Details on study schedule:
Satellite groups:
For the males and females in the 0 and 300 mg/kg bw/d groups, a 14-day recovery period was provided after administration for 42 days. The females in the recovery group were not subjected to mating.
Remarks:
Doses / Concentrations:
0, 30, 100, and 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Main groups: 12 males and 12 females (0, 30, 100, and 300 mg/kg bw/day)
Satellite groups: 5 males and 5 females (0, and 300 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were set based on the results of a 14-day repeated dose oral toxicity study of IAA in Crl:CD(SD) rats at 7 weeks of age at the start of administration (Dose levels: 15, 60, 250 and 1000 mg/kg bw/day, number of animals used: 3 males and 3 females per group). In that 14-day study, administration at 1000 mg/kg bw/day was associated with sedation of activity after dosing and death of 1 female each on day 6 and day 9 of administration, but there were no toxicological abnormalities in organ weight, haematological examination or blood chemistry examination in any surviving males or females. At 250 mg/kg bw/day and below dose levels, there were no toxicological abnormalities. Based on these results, 300 mg/kg bw/day, which is approximately 1/3 of the lethal dose level in the 14-day study and at which no deaths but effects by repeated administrations were expected, was selected as the high dose level and a total of 3 dose levels including 100 and 30 mg/kg bw/day which were calculated using a common ratio of approximately 3 were selected.

- Study design:
The dose levels were set at 30, 100 and 300 mg/kg bw, and a total of 4 groups, including a control group, were provided for the study. In the main group where animals were subjected to mating, each group consisted of 12 males and 12 females. In the recovery group where animals were not subjected to mating, 5 males and 5 females were assigned each to the control group and the high dose group. For the males in the recovery group, 5 males in the main group with animal numbers 08 to 12 in the last 2 digits were transferred after the day of final administration.

Satellite groups:
For the males and females in the 0 and 300 mg/kg bw/d groups, a 14-day recovery period was provided after administration for 42 days. The females in the recovery group were not subjected to mating.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times a day (prior to dosing, immediately after and approximately 2 hours after dosing) during the administration period and
once a day (in the morning) during the recovery period
- Observations: clinical signs, including abnormalities in external appearance, nutritional condition, posture, behavior and excrement

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the start of administration (all animals); once every week (males of the main and satellite groups, females of the satellite groups); once every week during the pre-mating administration period, and on the designated days (days 1, 7, 14 and 20 of gestation and day 4 of lactation) during the mating, gestation and lactation periods (females of the main groups).

BODY WEIGHT: Yes
- Time schedule for examinations:
- males of the main groups: 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration and on the day of necropsy;
- males and females of the satellite groups: 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration, on days 1, 4, 8, 11 and 14 of recovery and on the day of necropsy;
- females of the main groups: on days 1, 4, 8, 11, 15 (on days 18, 22 and 25 as well for uncopulated females) of administration, on days 0, 4, 7, 11, 14, 17 and 20 of gestation, and on days 0 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
- males of the main groups: on days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration;
- males and females of the satellite groups: on days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration and on days 1, 4, 8, 11 and 14 of recovery;
- females of the main groups: on days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20 of gestation and days 2 and 4 of lactation.

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Vaginal smears were collected from all females in the main group every day (in the morning) from the day after the start of administration to the day copulation was confirmed and examined microscopically.
During the pre-mating administration period, vaginal smear pictures were classified into proestrus, estrus, metestrus and diestrus and examined for the frequency of estrus and interval between estruses (estrous cycle; 4- or 5-day interval was regarded as normal).
During the mating period, vaginal smears were examined for the presence of sperm.
Sperm parameters (parental animals):
not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of liveborn pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Number of corpora lutea, implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively. Organ weights were determined for the 5 males and females/group that had been subjected to haematological examination: Brain, thyroid glands (including parathyroid glands), thymus, heart, liver, spleen, kidneys, adrenals, testes, and epididymides.

The following organs were preserved for microscopic examination:
Cerebrum, cerebellum, pituitary, spinal cord (thoracic), sciatic nerve, thyroid gland, parathyroid gland, adrenal, thymus, spleen, mandibular lymph node, mesenteric lymph node, heart, lungs (including bronchus), stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, urinary bladder, testis, epididymis, ovary, uterus, seminal vesicle, sternum (including bone marrow), femur (including bone marrow), macroscopic lesions, and the part for individual identification (ear auricle).
The organs/tissues of the 5 males and 5 females in the control group and the high dose group that were subjected to hematological/blood chemistry examinations and macroscopic lesions of all animals were subjected to microscopic examination.
Postmortem examinations (offspring):
GROSS EXAMINATION OF DEAD PUPS: yes
All liveborn pups were exsanguinated under ether anesthesia after measurement of body weight on day 4 of lactation, necropsied and examined for any abnormality in organs/tissues including those in the head, thorax and abdomen. Liveborn pups were individually weighed, and the average body weight per litter was calculated by sex.
Statistics:
Body weight, food consumption, water intake, number of estruses, estrous cycle, number of days until copulation, gestation period, number of corpora lutea, number of implantation sites, number of live pups, data of open field observations (number of defecations, number of rearings), manipulative test (landing foot splay), grip strength and motor activity, quantitative items of urinalysis, hematological examination, blood chemistry examination and organ weight data were tested by the appropriate method, i.e. Bartlett's test (two-tailed), F-test, Dunnett’s test, a Dunnett-type mean rank test, Student's t-test, Aspin-Welch's t-test.

For the implantation index, pre-implantation loss index, post-implantation loss index, stillbirth index, partrition index, live birth index, index of external abnormalities, and viability index, litter mean was calculated first and then analyzed by the appropriate method.

For the copulation index, insemination index, fertility index, gestation index, sex ratio of liveborn pups, auditory response, approach response, touch response, tail pinch response, pupillary reflex, aerial righting reflex, number of copulated animals in each group, number of males that impregnate females, number of pregnant females, number of females that had liveborn pups, number of male pups alive, and number of female pups alive, a total number of animals that showed such item was calculated and analyzed by chi-square test with Yates’ continuity correction (levels of significance: 0.05, 0.01, two-sided). If there were cells with expected frequency of not more than 5, the data were analyzed by Fisher’s exact test (levels of significance: 0.05, 0.01, two-sided).
Reproductive indices:
- Copulation index (%) = (Number of copulated animals / Number of mated animals) × 100
- Fertility index (%) = (Number of pregnant females / Number of copulated females) × 100
- Insemination index (%) = (Number of males inseminated females / Number of copulated
males) × 100
- Gestation period (days) = Number of days from day 0 of gestation to the day of delivery
- Delivery index (%) = (Number of females delivered live pups / Number of pregnant females) ×
100
- Implantation index (%) = (Number of implantation sites / Number of corpora lutea) × 100
- Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantation sites) /
Number of corpora lutea] × 100
- Post-implantation loss (%) = [(Number of implantation sites – Number of liveborn pups) /
Number of implantation sites] × 100
- Stillbirth index (%) = (Number of stillborn pups / Total number of pups born) × 100
- Parturition index (%) = (Number of stillborn and liveborn pups / Number of implantation sites)
× 100
- Live birth index (%) = (Number of liveborn pups / Total number of pups born) × 100
Offspring viability indices:
- Index of external abnormalities (%) = (Number of liveborn pups with external abnormalities /
Number of liveborn pups) × 100
- Sex ratio = [Number of male pups / (Number of male pups and female pups)] × 100
- Viability index on day 4 after birth = (Number of live pups on day 4 / Number of liveborn pups
on day 0) × 100
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Convulsion by contact stimulus was observed in a female in the 300 mg/kg bw group on day 9 of administration. Otherwise there was no abnormality in any animal in the main group or recovery group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the 300 mg/kg bw group, body weight gain during the administration period was low in males in the main group and the difference was statistically significant. The body weight was transiently low and the difference was statistically significant on day 39 of administration. During the recovery period, body weight gain of males in the 300 mg/kg bw group tended to be higher than that of the control group though it was not statistically significant. Otherwise there was no test article-related effect in males or females in the main group or recovery group.
There were no test article-related effects in males or females in the main group or recovery group. Significantly low values were observed in males in the 300 mg/kg group on day 39 of administration and in females in the 100 mg/kg bw group on day 8 of administration; however, they were judged to be incidental since they were transient changes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no animals showing abnormal estrous cycles, and there were no significant differences in the average length of the estrous cycle between the control group and any treatment group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Copulation was observed in almost all couples by day 5 after the start of mating, and it was observed in all couples by day 13. All females were pregnant except 1 female in the 300 mg/kg bw group. There were no significant differences between the control group and any test article administration group in the copulation index, insemination index or fertility index.
In the delivery status, all animals had normal delivery on day 21 or 22 of gestation, and there were no significant differences between the control group and any test article administration group in the delivery index, gestation period, number of corpora lutea, number of implantation sites, implantation index, pre-implantation loss (%), post-implantation loss (%), stillbirth index, parturition index, number of liveborn pups or live birth index.
In the lactation condition, no dams had abnormalities in nesting or lactational behavior.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At the end of the administration period, decreases in the absolute and relative weights of the liver were observed in males in each administration group and they were statistically significant.
Otherwise significant differences from the control group were observed in the following organs: adrenal, brain, heart. However, they were judged to be incidental since they were mild changes only in the absolute weight, and there were no test article-related changes in the relative weight or histopathological examination.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At the end of the administration period: Some changes were observed in the following organs/tissues: liver, lung, stomach. At the end of the recovery period some changes were observed in the following organs/tissues: stomach, testis. However, they were thought to be incidental based on the incidence of their occurrences and pathological nature.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Some changes were observed; however, they were thought to be incidental based on the incidence of their occurrences and histopathological nature.
For further details, please refer to 7.5.1 Repeated dose toxicity: oral.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproduction up to the highest dose.
VIABILITY (OFFSPRING)
The number of pups that died during the lactation period was 18 in the control group, and 0, 2 and 1 in the 30, 100 and 300 mg/kg bw groups, respectively. The viability index on day 4 of lactation was significantly high in the 30 mg/kg bw group, but it was due to a good viability index (100%).

CLINICAL SIGNS (OFFSPRING): liveborn pups
There were no significant differences in the sex ratio or body weight at the time of birth between the control group and any treatment group, and there were no external abnormalities.

BODY WEIGHT (OFFSPRING)
There were no significant differences in the body weight at the time of birth or on day 4 of lactation between the control group and any test article group.

GROSS PATHOLOGY (OFFSPRING)
Thymic remnant in the neck was observed in 2 males in the control group, 2 males and 1 female in the 30 mg/kg bw group and 1 male in the 300 mg/kg bw group, and pelvic dilatation (bilateral) in 1 male in the 100 mg/kg bw group; however, these changes were considered to be incidental since they are known to occur spontaneously in rats and the incidence of their occurrence was small.
There were no other abnormalities in liveborn pups.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects in liveborn pups up tp the highest dose.
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Several data were used in a weight of evidence approach for the assessment of developmental toxicity. The whole database was considered reliable and suffcient for assessment.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

3-methylbutan-1-ol was tested in two Combined 28-Day Repeated Dose Toxicity Studies with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and in compliance with GLP regulations (Kuraray Co. Ltd. 2008 and BASF 2018). In the older study, the substance was administered to male and female Sprague-Dawley strain SPF rats by gavage at dose levels of 0 (control group), 30, 100 or 300 mg/kg bw for a total of 42 days to males (for 14 days before mating throughout the mating period up to the day before necropsy) and for a total of 41 to 53 days to females (for 14 days before mating throughout the mating and gestation periods up to day 4 of lactation) to examine its repeated dose toxicity and reproductive and developmental toxicity. For the males and females in the 0 and 300 mg/kg groups, a 14-day recovery period was provided after administration for 42 days to examine reversibility of the toxic changes. The females in the recovery group were not subjected to mating. There were no test article-related effects on estrous cycle, number of days until copulation, copulation index, insemination index or fertility index. There were no test article-related effects on delivery index, length of gestation period, number of corpora lutea, number of implantation sites, implantation index, index of pre-implantation loss, index of post-implantation loss, index of stillborn pups, parturition index, number of liveborn pups, live birth index or sex ratio, and there were no abnormalities in the lactation condition during the lactation period. In live born pups, there were no test article-related changes in body weight, external observation, gross pathological findings or viability index on day 4 of lactation. Based on the results described above, it was judged that the no adverse effect levels for reproductive and developmental toxicity in male and female parent animals and pups were 300 mg/kg bw/day.

To resolve remaining uncertainities about the test substances' toxicity up to limit doses, in the more recently performed GLP-compliant OECD 422 study, the test item was administered daily as addition to the drinking water in different concentrations to groups of 10 male and 10 female Wistar rats to screen for potential systemic, reproductive and developmental toxicity with doses up to 1000 mg/kg bw (limit dose). After a two-week premating period, these parental animals were paired and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or PND 13. In males the overall mean dose of the test substance throughout the study was approx. 71 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 229 mg/kg bw/d in the 3750 ppm group and approx. 785 mg/kg bw/d in the 12500 ppm group; in females it was approx. 116 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 359 mg/kg bw/d in the 3750 ppm group and approx. 1273 mg/kg bw/d in the 12500 ppm group.

Analyses confirmed the overall accuracy of the prepared concentrations in the drinking water. The stability of these preparations was also demonstrated over a period of 10 days under ambient conditions.

In the clinical examinations of the F0 parental animals no clinical symptoms were caused by the test compound up to the high-concentration of 12500 ppm. In the in-depth investigations including the detailed clinical observation, the functional observational battery and the measurement of motor activity no treatment-related differences to control were observed at any concentration. Water consumption, food consumption and body weights / body weight gain did not show important test substance-related changes.

Concerning clinical pathology (including thyroid hormone measurement) no treatment-related, adverse effects were observed up to a concentration of the compound of 12500 ppm in the drinking water.

Regarding pathology, there were no treatment-related organ weight changes, gross lesions, and histopathological findings in male and female Wistar rats. Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups (1250, 3750, and 12500 ppm) during the entire study. Most F0 parental animals across all test groups proved to be fertile and those individuals failing to generate offspring did not show any specific gross or histopathological findings. Mating behavior, conception, implantation and parturition were not influenced. Pups showed normal development up to scheduled sacrifice. Neither determination of anogenital distance/index not the count of nipple/areola anlagen revealed any treatment-related changes up to and including a concentration of the test item of 12500 ppm in the drinking water.

Furthermore, the read across substance reaction mass of 2-methylbutan-1-ol and pentan-1-ol was tested in an GLP-compliant OECD 422 study (BASF 2018). It

was administered daily as addition to the drinking water in different concentrations to groups of 10 male and 10 female Wistar rats to screen for potential systemic, reproductive and developmental toxicity. After a two-week premating period, these parental animals were paired and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or PND 13. In males the overall mean dose of the test substance throughout the study was approx. 77 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 254 mg/kg bw/d in the 3750 ppm group and approx. 842 mg/kg bw/d in the 12500 ppm group; in females it was approx. 117 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 372 mg/kg bw/d in the 3750 ppm group and approx. 1239 mg/kg bw/d in the 12500 ppm group.

Analyses confirmed the overall accuracy of the prepared concentrations in the drinking water. The stability of these preparations was also demonstrated over a period of 10 days under ambient conditions.

In the clinical examinations of the F0 parental animals no clinical symptoms were caused by the test compound up to the high-concentration of 12500 ppm. In the in-depth investigations including the detailed clinical observation, the functional observational battery and the measurement of motor activity no treatment-related differences to control were observed at any concentration. Water consumption, food consumption and body weights / body weight gain did not show important test substance-related changes. A small decrease in food consumption of high-dose males as well as temporary increase of water consumption in high-dose females were not accompanied by any body weight changes or other clinical findings and thus not noteworthy enough to be considered adverse.

Concerning clinical pathology (including thyroid hormone measurement) no treatment-related, adverse effects were observed up to a concentration of the compound of 12500 ppm in the drinking water.

Regarding pathology, there were no treatment-related organ weight changes, gross lesions, and histopathological findings in male and female Wistar rats. Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups (1250, 3750, and 12500 ppm) during the entire study. Most F0 parental animals across all test groups proved to be fertile and those individuals failing to generate offspring did not show any specific gross or histopathological findings. There were no test article-related effects on estrous cycle, number of days until copulation, copulation index, insemination index, or fertility index. There were no test article-related effects on delivery index, length of gestation period, number of corpora lutea, number of implantation sites, implantation index, index of pre-implantation loss, index of post-implantation loss, index of stillborn pups, parturition index, number of liveborn pups, live birth index or sex ratio, and there were no abnormalities in the lactation condition during the lactation period. In live born pups, there were no test article-related changes in body weight, external observation, gross pathological findings or viability index on day 4 of lactation. Neither determination of anogenital distance/index not the count of nipple/areola anlagen revealed any treatment-related changes up to and including a concentration of the test item of 12500 ppm in the drinking water. This results in a calculated NOAEL of 1000 mg/kg bw for the assessment of adverse effects on reproduction at the screening level.

In addition, repeated dose toxicity was investigated in a 90 day drinking water study performed according to OECD guideline 408 with 3-methylbutan-1-ol (BG-Chemie 1990). Ten Wistar rats per sex and dose received nominal doses of 80, 340 and 1250 mg/kg bw/day 3-methylbutan-1-ol in the drinking water for 3 months. At the end of the 3-month administration period all animals were sacrificed by decapitation and were assessed by gross pathology. Subsequently, a histopathological examination was carried out including the reproductive organs. Regarding toxicity to reproductive organs, no abnormalities were found by histological analysis. Thus, the highest dose level tested, 1250 mg/kg bw /day, was the NOAEL under the conditions of the study.

These results were supported by a publication describing a subchronic study performed with pentan-1-ol. In a subchronic repeated dose toxicity study conducted equivalent to OECD guideline 408, 15 ASH/CSE rats per sex and dose received doses of 50, 150 and 1000 mg/kg bw/day pentan-1-ol in corn oil by gavage for 13 weeks (Butterworth et al. 1978). At necropsy all tissues were examined for gross abnormalities including the reproductive organs. No abnormalities in the reproductive organs were found by histological analysis. Therefore, the highest dose of 1000 mg/kg bw can be seen as the NOAEL regarding reproductive toxicity of pentan-1-ol. A detailed read-across justification is attached in IUCLID chapter 13. 

Taken together, there are no hints for a reproductive toxic potential of 3-methylbutan-1-ol, nor was any relevant systemic toxicity observed up to at least 1000 mg/kg subchronic exposure for the substance itself or its structural analogue. The available experimental data are sufficient to assess the potential toxicity to reproduction by 3-methylbutan-1-ol.

Effects on developmental toxicity

Description of key information

Toxicity to reproduction (development):

- inhalative: NOAEC = 10 mg/L air (developmental toxicity) = sat. vapour conc. (rat and rabbit, OECD 414)
- inhalative: NOAEC = 14 mg/L air (maternal and developmental) = sat. vapour conc. (Sprague-Dawley rat, vapour inhalation, GD 1-19) - RA to 71-41-0

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Remarks:
BASF AG, Department of Toxicology
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-7950 Biberach/Riss, FRG). The animals were free from any clinically evident signs prior to the beginning of the study.
- Age at study initiation: ca 11 weeks
- Weight at study initiation: ca 216 g
- Housing: singly in wire cages (type D III of Becker & Co, Castrop-Rauxel, FRG)
- Diet (e.g. ad libitum): KLIBA rat/mouse laboratory diet 24-343-4 10 mm pellets, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; during the exposure-free observation period
- Water (e.g. ad libitum): tap water; during the exposure-free observation period
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS in fully air-conditioned rooms
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the animals were exposed singly in wire cages (D III) in glass-steel inhalation chambers (manufacturer: BASF Aktiengesellschaft), Volume Vz 1,100 1 (test group 1, 2 and 3), Volume V 2 1,600 1 (test group 0 and parts of the test groups during the preflow period).
- Method of holding animals in test chamber: whole-body exposure system (glass-steel inhalation chamber) with a volume of about 1.1 m3 (test groups 1 - 3); volume of the inhalation chamber of the control group: 1.6 m3
- Source and rate of air: the test substance was supplied by means of two continuously driven piston pumps (Unita, Braun) in test group 1, a continuously metering pump (Optimat MP) in test group 2, and another continuously metering pump (Desaga) in test group 3 to a vaporizer heated with a circulating thermostat and evaporated. The evaporation temperatures are shown in the following table.

_______________________________________________________________________________
Test group /ml/hour /Evaporation temperature (°C) /Supply air (l/hour) /Exhaust air (l/hour)
-----------------------------------------------------------------------------------------------------------------------
0 /Fresh air /- /30000 /29500
1 /13.7 - 14.3 /50 /21500 /22000
2 /75.6 - 82.8 /60 /21500 /22000
3 /295 - 305 /70 /21500 /22000
_______________________________________________________________________________

A stream of fresh air measured with a rotameter took up the vapors. A further stream of fresh air was passed in downstream of the vaporizer. After passing through a mixing device, this mixture of vapors and air was supplied to exposure system

- Temperature, humidity, pressure in air chamber: the pressure in the inhalation chambers was measured continuously (inclined manometer) and recorded, as a rule, 3 times/exposure. Conditioned supply air ( about 50% humidity, 22 °C) was used for the exposure in all test groups. The temperature in the exposure systems was measured continuously (digital thermometer, Diehl) and recorded, as a rule, 3 times/exposure. The relative humidity in the chambers was checked with a humidity measuring probe (Vaisala) at least once a day and also recorded.
- Air flow rate: all air flows, supply air and exhaust air were adjusted by means of flowmeters (rotameter) for all test groups and recorded, as a rule, 6 times/exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: the concentration in the inhalation chambers was monitored by means of a GC-method. The concentrations of the test groups were analyzed by gas chromatography after absorption of MEB samples in 2-propanol.
The gas chromatographs were calibrated with weighed amounts of the test substance.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration in the inhalation chambers was monitored by means of a GC-method.
The concentrations of the test groups were analyzed by gas chromatography after absorption of MEB samples in 2-propanol.
The gas chromatographs were calibrated with weighed amounts of the test substance.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/4
- Length of cohabitation: 15.5 hours (overnight)
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6 - 15 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
20 days
Remarks:
Doses / Concentrations:
0.51±0.015, 2.50±0.169 and 9.8±0.66 mg/l
Basis:
analytical conc.
gas chromatograph monitoring
Remarks:
Doses / Concentrations:
0.50, 2.50 and 10.0 mg/l
Basis:
nominal conc.
target concentration
No. of animals per sex per dose:
25 (in driplicate in each group)
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: in a pretest no maternal toxic effects could be observed at concentrations up to 5 mg/l, which is the limit test concentration. Because fetotoxic effects were reported with other alcohols at somewhat higher concentrations, the highest concentration selected for the study was 10 mg/l, which is near to the saturated vapor concentration at approx. 20°C (12 - 14 mg/l). In order to determine dose-response relationships, an intermediate (2.5 mg/ml) and a low (0.5 mg/ml) concentration levels were also selected.
Details on the range finding study are included in the IUCLID dossier as a seperate supporting study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: the behavior and state of health of the test animals were checked on workdays at least 3 times on exposure days and, as a rule, once during the post-exposure observation period.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of the animals was checked on day 0 day of dectection of sperm) and on days 3, 6, 9, 12, 15, 18 and 20 p.c. As a rule, the animals were weighed at the same time of the day. The difference between the body weight on the day of weighing and the body weight on the previous weighing was calculated. Moreover the same was done for 3 different study periods:
* preflow period (day 0- 6 p.c.)
* exposure period (days 6 - 15 p.c.)
* observation period (days 15 - 20 p.c.).
These values are defined as body weight change. Moreover, the corrected body weight gain (body weight on day 20 p.c. minus the body weight on day 6 p.c. minus weight of the uterus before it was opened) was determined after the dams had been sacrificed at the end of the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: on day 20 p.c. the dams (as well as moribund dams) were sacrificed by cercical dislocation and the fetuses removed by cesarean section. These animals and dams which died intercurrently as well as the contents of uterus from these animals were investigated, if possible in the same way as at terminal sacrifice (exception: uterus weight).
- Organs examined: after the dams had been sacrificed, they were necropsied and assessed by gross pathology. The uterus and the ovaries were removed and the following data were recorded: weight of uterus before it was opened, number of corpora lutea, number and distribution of implantation sites classified as live fetuses or dead implantations: early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy); late resorptions (embryonic or fetal tissue in addition to placental tissue visible); dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened). Furthermore, calculations of conception rate and pre and postimplantation losses were carried out.

OTHER: a check for dead animals was made daily.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations:Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
The statistical evaluation of the data was carried out on the computer systems of the Department of Toxicology (Dr. Hoffmann responsible). Examinations of the dams and fetuses Dunnett's Test (8 - 9) was used for statistical evaluation of body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre and postimplantation loss, resorptions and live fetuses. Fisher's Exact Test ( 10) was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
Significances resulting from these tests have been indicated in the tables (a for p < 0.05, b for p < 0.01).
Indices:
- The conception rate (in 3;) was calculated according to the following formula: (number of pregnant animals/ number of fertilized animals)*100
- The preimplantation loss (in %) was calculated according to the following formula: (number of corpora lutea - number of implantations/number of corpora lutea)*100
- The postimplantation loss (in %) was calculated from the following formula: (number of implantations - number of live fetuses/number of implantations)*100
Historical control data:
Historical control studies from Himalayan rabbits carried out in BASF's Department of Toxicology between 1986 and 1990
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Only transient impairment in the body weight change of the dams was observed at the beginning of the exposure period (days 6 - 9 p.c.).
Dose descriptor:
NOAEC
Effect level:
2.5 mg/L air (nominal)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The fetuses did not show any substance-related effects (neither embryo-/fetotoxic nor teratogenic effects).
Dose descriptor:
NOAEC
Effect level:
10 mg/L air (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

1) Examinations of the dams:

- Clinical Examinations:

Only pregnant dams were used for the calculations of mean maternal body weights and body weight change. Only pregnant dams with scheduled sacrifice (day 20 p.c.) were taken for the calculation of mean gravid uterine weights, mean net maternal body weight change (corrected body weight gain) and summary of reproduction data.

In this study 3, 4 and 2 females (respectively in test group 1, 4 and 3) were excluded from the above mentioned calculations since they did not conceive (while one animal died in the test group 2).

*Clinical signs and findings: there were no substance-induced clinical signs or findings in all test groups (0 - 3) at any time of the study period (preflow, exposure, post-exposure observation).

*Lethality: no deaths were recorded throughout the study period in test groups 0, 2 and 3. One animal of test group 1, which was not pregnant, was found dead in cage on day 12 p.c.

 

- Body weight:

The body weights of all animals in test group 1 compared to the control (0) were not statistically significant. In test group 2, the body weight change was statistically significantly increased (p < 0.05) between days 12 -15 p.c. In test group 3, compared to the control (group 0), the body weight change was statistically significantly decreased (p < 0.05) between days 6 -9 p.c. and statistically significantly increased (p < 0.01) between day 12-15 p.c.

Over the total exposure period (days 6-15 p.c.) no substance-related effects were observed. It cannot be decided clearly, whether there was a slight effect on body weight retardation in the first days of exposure (6 - 9 p.c.) followed by an adaptation/recovery phase in the further days of exposure (12 - 15 p.c.) or whether this is an incidental finding. In case this effect may be considered as a very slight indication of maternal toxicity only during the first phase of exposure to a very high concentration of 10 mg/l of test substance.

 

- Body weight change:

The results of the corrected body weight gain (body weight on day 20 p.c. minus body weight on day 6 p.c. minus weight of the uterus before it was opened) do not show any differences of biological relevance between the groups.

 

- Examinations of the dams at termination

*Necropsy findings: there were no substance-related observations at necropsy in any of the dams.

*Uterus weight: the uterus weights of the animals were not influenced by the exposure to the test substance.

*Reproduction data of dams: the conception rate varied between 80 and 100%. There were no substance-related and/or statistically significant differences between the groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age (also compared to the lab's historical control data).

 

2) Examinations of the fetuses

- Sex distribution of fetuses:

The sex distribution of the fetuses in test groups 1- 3 (0.5, 2.5 and 10 mg/l) was comparable with the control fetuses (the differences observed in comparison being without any biological relevance).

 

- Weight of placentae:

The mean placental weights in test groups 1-3 (0.5, 2.5 and 10 mg/l) were not influenced by the administration of the test substance to the dams (the differences observed being without any biological relevance).

 

- Weight of fetuses:

The mean fetal weights were not influenced by the test substance exposure (all values are within the range of biological variation (also comparable to the historical control values).

 

- External examination of the fetuses:

The external examination of the fetuses revealed only one malformation (polydactyly) in one fetus out of 326 fetuses in the highest dose group (10 mg/l) and no variations in any group.

So-called unclassified observations were recorded for 4 fetuses of the control group (blood coagulum around placenta), 9 fetuses (from one litter) in test group 2 (2.5 mg/l) (placentae necrobiotic) and 1 fetus of the highest dose group (10 mg/l) (placentae fused).

 

- Soft tissue examinations of the fetuses:

The examination of the organs of the fetuses revealed two malformations in test group 2 (2.5 mg/l). For one fetus a globular shaped heart, for another one dextrocardia was recorded. Variations were detected in all groups. The very common finding (dilated renal pelvis) in the rat strain used in this study occurred without any dose-response relationship. The occurrence of the other variation (hydroureter) did not show any statistically significant difference between the groups. The examination of the organs of the fetuses revealed no so-called unclassified observations (like blood coagulum around the bladder) in any test group.

 

- Skeletal examination of the fetuses:

Various malformations of the sternebrae (sternebra(e) bipartite, ossification centers dislocated, cleft sternum) and/or the vertebral column (e.g. thoracic vertebral body/bodies dumbbell-shaped (asym.) or bipartite (asym.)) were seen in very few fetuses (4 - 8) in all test groups, the differences not being statistically significant. The variations elicited were related to the ribs shortened or missing 13th, accessory 14th ribs or rudimentary cervical ribs) and the sternum (sternebra(e) of irregular shape, bipartite or accessory sternebra) and were found in all groups to about the same extent with the exception of a lower incidence of shortened 13thrib(s) in the highest dose group.

In all groups signs of retardations (incomplete or missing ossification of hyoid, skull, metacarpal or metatarsal bones, vertebral bodies and/or sternebra(e)) were found without any clear differences of biological relevance between the groups.

 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
10 000 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Several data were used in a weight of evidence approach for the assessment of developmental toxicity. The whole database was considered reliable and sufficient for assessment.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity of 3-methylbutan-1-ol was investigated in a prenatal developmental study performed according to OECD test guideline 414 (BG-Chemie 1990). 25 Wistar rats were exposed to test substance vapours at 0.5, 2.5 and 10 mg/L air for 6 hours/day on days 6 - 15 of gestation. The number of corpora lutea, implantations, resorption sites, and live fetuses per sex was recorded. Fetuses were examined for skeletal malformations or visceral abnormalities. As a result, a marginal and transient (days 6-9) retardation of body weight gain was observed in the dams of the highest dose group, while the fetuses did not show any embryo-/fetotoxic or teratogenic effects in all dose groups. Thus, the NOAEC was 2.5 mg/L air for maternal toxicity and 10 mg/L air for developmental toxicity, respectively.

In a similar study also performed according to OECD TG 414, Himalayan rabbits as second species were exposed to 3-methylbutan-1-ol substance vapours at 0.5, 2.5 and 10 mg/L air, 6 hours/day, on days 7 - 19 of gestation (BG-Chemie 1990). Animals were sacrificed on gestation day 29. Gravid uterus weight, number of copora lutea, implantation sites, early and late resorptions and live foetuses were recorded. All foetuses were examined for visceral and skeletal changes, including a head examination. The only effect was a decreased body weight in the high dose animals between gestation days 7 and 10, comparable to the findings in rats. In addition, signs of beginning eye irritation were observed in this group. Thus, the NOAEC was 2.5 mg/L air for maternal toxicity and 10 mg/L air for developmental toxicity in rabbits.

Developmental toxicity of pentan-1-ol was examined in a study, where 15 female, sperm-positive Sprague-Dawley rats were exposed to a concentration of 14 mg/L air, the highest vapour concentration technically achievable (Nelson et al. 1989). The exposure duration was 7 hour/day on days 1 - 19 of gestation. At the end of the exposure period, the dams were sacrificed and ovaries and uterine content as well as fetuses were examined. The number of corpora lutea, implantations, resorption sites and live fetuses was recorded. One-half of the fetuses were examined for skeletal malformations, while the remaining fetuses were examined for visceral abnormalities. At 14 mg/L air, no overt maternal toxicity was observed. Overall feed consumption was lower than in controls, but water consumption remained unchanged. Although the weight gain was slightly decreased, this effect did not reach statistical significance. The number of corpora lutea, resorptions, gender ratio and fetal weights were also not affected by treatment. Although small reversible delays in ossification of the caudal vertebrae, the sternum, the metacarpals and the hind paw phalanges were reported, these effects were not statistically significant. In addition, no malformations of the fetuses were observed. Thus, the dose of 14 mg/L air can be seen as the NOAEC regarding maternal as well as developmental toxicity of pentan-1-ol.

Thus, no indications of a developmental toxic or teratogenic effect were seen in animal studies with 3-methylbutan-1-ol and its structural analogue pentan-1-ol. A detailed justification for this read-across is attached in IUCLID chapter 13

Justification for classification or non-classification

The available data are considered reliable and suitable for classification purposes under Regulation (EC) No 1272/2008 (CLP). As a result, no classification for toxicity to reproduction or developmental toxicity under Regulation (EC) No 1272/2008 is required for 3 -methylbutan-1 -ol.