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), an extended one generation study (OECD 443) and in subchronic oral toxicity studies.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Feb 2018 - Oct 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
Jul 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
Study design based on ECHA final decision CCH-D-2114346807-40-01/F
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: BASF SE
- Batch No.of test material: 59086375L0
- Expiration date of the lot/batch: 17 Jan 2019
- Purity: 99.7%
- Appearance: Liquid/colorless clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: guaranteed and verified
- Homogeneity: given

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The required quantities of test substance, depending on the dose group, were weighed into appropriate beakers. A vessel with a capacity of 30L (equipped with a with magnetic rod) was tared to 0g. Then 10L of drinking water were added. The test substance was completely transferred from the beaker into the vessel, the beakers rinsed with further amounts of drinking water. Afterwards an appropriate amount of drinking water was added to obtain the desired concentrations for the different dose groups. These preparations were stirred until the test substance was completely dissolved.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to test guidelines.
This strain was selected since extensive historical control data were available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 34 (+/-1) days; (F1) 3 wks
- Weight at study initiation: females (mean) 110 g; males (mean) 108.5 g
- Fasting period before study: no
- Housing: five per cage, females after weaning and males postmating: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6 am to 6 pm)

IN-LIFE DATES: From: 20 Feb 2018 To: F1: 26 - 30 Jun and 02 Jul 2018; F0M: 12 - 14 Jun 2018
F0F: 14 May and 3 - 4 Jul 2018; 1A: 31 Aug and 03 - 05 Sep 2018, 1B: 29 - 30 Aug and 6 - 7 Sep 2018
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): continuous
- Mixing appropriate amounts with (Type of food): drinking water

VEHICLE
- water
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max 2 weeks; about 16.00 h until 6.30 - 9.00 h of the following morning.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): group
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were carried out at the beginning and toward the end of the premating phase, in female drinking water once in the lactation period as well as for cohort 1A and 1B animals toward the end of the postweaning period.
Duration of treatment / exposure:
F0 males: 10 weeks pre-mating, 2 weeks mating, 3 weeks post-mating
F0 females: 10 weeks pre-mating, 2 weeks mating, pregnancy and lactation

F1: In-utero, pre-weaning, post-weaning and cohort 1A and 1B 10 weeks post-weaning
Frequency of treatment:
daily
Details on study schedule:
100 males and females were treated for a minimum of 10 weeks, then males and females from the same dose group were mated, overnight at a ratio of 1:1. Females were allowed to deliver pups until PND 4 or PND 21 or PND 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts which were subjected to specific postweaning examinations. On PND 4 blood samples were collected from a maximum of 10 surplus (culled) F1 pups per sex and group. On PND 22 blood samples were collected from 10 surplus F1 pups per sex and group.
Blood samples were taken from 10 animals per test group of the F0 parental animals and cohort 1A animals.
Shortly before weaning of the F1 pups the F0 generation parental male animals were sacrificed.
After weaning of F1 pups the F0 generation parental female animals were sacrificed.
Dose / conc.:
1 250 ppm
Dose / conc.:
3 750 ppm
Dose / conc.:
12 500 ppm
No. of animals per sex per dose:
25 (F0), 20 (F1A), 25 (F1B)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were selected based on a previously conducted screening OECD guideline 422 study with 3-Methyl-1-butanol (BASF 2018). In this present study, a NOAEL for general systemic toxicity was observed around the limit dose (1000 mg/kg bw). Therefore a similar high-dose was chosen.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: not specified
- Other: During the lactation period the test substance concentrations in the drinking water of the F0 females were reduced to 50%. This drinking water adjustment derived from historical body weight and water consumption data maintained the dams at the desired target doses of the test substance during this period of increased water intake.
Positive control:
not included
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: once a week, except during pregnancy and lactation; GD 0-4, 4-7, 7-10, 10-14, 14-17 and 17-20 and PND 1-4, 4-7, 7-10, 10-14, 14-17, 17-20 and 20-21, respectively.

FOOD CONSUMPTION:
- Time schedule: once a week, except: not determined after the 10th premating week (male F0 animals) and during the mating period, During pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0-7, 7-14 and 14-20, During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Over a period of 3 days, except during pregnancy and lactation GD 0-1, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21 and PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21, respectively.

CLINICAL PATHOLOGY
- Time schedule: At termination from 10 F0 parental and cohort 1A males and females

HEMATOLOGY: Yes
Parameters analyzed: Leukocyte count (WBC), Erythrocyte count (RBC). Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time

CLINICAL CHEMISTRY: Yes
Parameters analyzed: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), -Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Total thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: Yes
Parameters analyzed: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity (SP.GR.) [g/L], Sediment, Color turbidity (COL, TURB), Volume (VOL) [mL]
Oestrous cyclicity (parental animals):
Evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75.
Sperm parameters (parental animals):
Parameters examined in all F0 male parental and cohort 1A male generations: sperm head count (testis), sperm head count (cauda epididymis), sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

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

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals of F0 and cohort F1A
- Maternal animals: All surviving animals, F0: post-weaning

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

HISTOPATHOLOGY
According to table 1 (any other information on materials and methods)

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides (left)
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes (left)
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighted together (left and right).

The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix uteri
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens

- Differential Ovarian Follicle Count (DOFC)in F1 generation: Both ovaries from each female (test groups 10 and 13) were embedded in paraffin blocks.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was sacrificed at PND 4 or PND 22, cohort 1A and 1B animals were dosed for 10 further weeks post-weaning

GROSS NECROPSY
- All cohort 1B animals were sacrificed by decapitation under isoflurane anesthesia.

ORGAN WEIGHTS:
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Cauda epididymis
3. Epididymides
4. Liver
5. Ovaries
6. Pituitary gland
7. Prostate
8. Testes
9. Seminal vesicles including coagulating gland
10. Uterus (with cervix)

Organ/Tissue fixation
The following organs or tissues were fixed in 4% formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymides (fixed in modified Davidson ´s solution)
6. Liver
7. Ovaries (fixed in modified Davidson´s solution)
8. Pituitary gland
9. Prostate
10. Seminal vesicles
11. Testes (fixed in modified Davidson ´s solution)
12. Uterus
13. Vagina

HISTOPATHOLOGY:
All organs listed above were preserved in adequate fixative. Histopathological processing and examination by light microscopy was not performed.

Pathological examinations of surplus F1 generation pups on PND 22 were sacrificed and organ weights (anesthetized animals final body weight, brain, spleen, thymus) and organ/tissue fixation (all gross lesions, brainn, mammary gland of male and female, spleen, thymus, thyroid glands) was performed. No histopathology performed.
Statistics:
- Dunnett's test (two-sided): Water consumption, food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), estrous cycle duration, number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter, duration of sexual maturation (days to vaginal opening, days to preputial separation), anogenital distance, anogenital index
- Fisher's exact test: Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy, sexual maturation data (vaginal opening, preputial separation)
- Wilcoxon test: Presence of areolae/nipples, proportions of affected pups per litter with necropsy observations, blood parameter, Urinalysis, Urine pH volume and specific gravity, DOFC, weight parameteres
- Kruskal-Wallis test: Number of cycles and cycle length, blood parameter, Urine pH volume and specific gravity, Spermanalysis, weight parameters
Reproductive indices:
Male mating index, Male fertility index, female mating index, female fertility index, gestation index, live birth index, postimplantation loss
Offspring viability indices:
Viability index, Lactation ondex, sex ratio, anogenital index
Clinical signs:
no effects observed
Description (incidence and severity):
Two females (Nos. 118 and 121 - 0 ppm) of the control, two females (Nos. 130 and 147 - 1250 ppm) of test group 01 and one female (No. 169 - 3750 ppm) of test group 02 did not deliver F1 pups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
12500 ppm: In males, body weights were below control values from premating day 28 onwards until the end of the study, most of the time the difference was statistically significant. The average final inlife weight of these males was 7% below control. Accordingly, body weight gain was reduced.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A temporary reduction of food consumption of the high-dose F0 females during premating days 0 - 7 (about 6%) was considered to be a marginal change which had no impact on the overall food consumption or body weight gain of these females. Thus, it was considered to be of no toxicological relevance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In parental males and females (test groups 1, 2 and 3; 1250, 3750 and 12500 ppm) no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
- Male mating index: control, 1250, 3750, 12500 ppm were 96%, 96%, 100% and 100%, resp.
- Male fertility index: ranged between 92% and 100%, no effects observed
- Female mating index: control, 1250, 3750, 12500 ppm were 96%, 96%, 100% and 100%, resp.
- Female fertility index: ranged between 96% and 100%, no effects observed
- Gestation index: 100% in all test groups
- Implantation: not affected
- No indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences
Dose descriptor:
NOAEL
Effect level:
405 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
1 521 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects
Dose descriptor:
NOAEL
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other:
Remarks:
about 1221 mg/kg bw/d in males and 1521 mg/kg bw/d in females),
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
F1 PUPS
- Live birth index: 100% in all test groups
- Viability index: 99%, 98%, 100%, 99% in control, 1250, 3750, 12500 ppm

COHORTS
F1A, F1B: no mortalities
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS
- no effects observed

COHORTS
- F1A: The statistically significantly decreased body weight change in the low-dose male animals during study days 35 - 42 was considered to be spontaneous in nature.
- F1B: The body weights of the high-dose males were statistically significantly below the control values on study day 14 (about 6%) and the body weight change was statistically significantly below the control values during study days 7 - 14 (about 13%). This temporary decrease had no impact on the overall body weight gain of these males. Thus, it was considered to be of no toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
COHORTS
- F1A: In males of the 12500 ppm group, food consumption statistically significantly below control values during study days 35-42 (about 9%). This temporary decrease had no impact on the overall food consumption or body weight gain of these males. Thus, it was considered to be of no toxicological relevance.
- F1B: 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):
COHORTS
- F1A: The statistically significantly increased water consumption in the low- and mid-dose female animals during several parts of the study was considered to be spontaneous in nature.
- F1B: Water consumption of the high-dose males was statistically significantly below the concurrent control values during study days 10 - 14 (about 22%).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: In females of 1250 ppm, relative eosinophil cell counts were significantly increased, but this change was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: In males of 1250 and 12500 ppm, triglyceride values were significantly decreased. The change in test group 11 was not dose-dependent. The values in 12500 ppm group were within the historical control range (males, triglycerides 0.61-1.37 mmol/L). In females of group 3750 ppm, urea levels were significantly decreased, but the values were not dose-dependently altered. Therefore, these changes were regarded as incidental and not treatment-related.
At PND22, in males of group 3750 ppm, T4 values were significantly increased, but the change was not dose-dependent. Therefore, this change was regarded as incidental and not treatment-related.
COHORTS
- In all males and females of the F1A cohort, no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
F1 PUPS: no effects observed.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Incidental entry of test group 12500 ppm into female puberty (small average difference to the control of about one day, apparent delay was solely due to 4 high-dose individuals entering puberty after the age of 36 days). Preputial separation comparable across all test groups.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
F1 PUPS: no effects observed. Anogenital index comparable to control
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
F1 PUPS: no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS
- F1 weanlings not selected for cohorts on PND 22: no effects

COHORTS
- F1A: When compared the control group (set to 100%) with test groups 1250, 3750 and 12500 ppm, the mean absolute weights of the thymus of female animals were significantly changed (86%, 89% and 87%).
When compared the test control group (set to 100%) with test groups 1250, 3750 and 12500 ppm the mean relative weights of the liver and thymus were significantly changed (statistically significant changes with an asterisk):
Liver (male animals): 95%*, 96%* and 96%*
Thymus (female animals): 86%**, 89%, and 87%*
The absolute thymus weight of the control group were comparably high and the absolute thymus weight of females and the absolute liver weight in males were within historical control values. Therefore, these weight changes were regarded to be incidental and unrelated to treatment.
- F1B: The decrease of the absolute weight of the seminal vesicles was the only parameter changed. The relative weight was not significantly changed. It was therefore not regarded to be a treatment-related effect. Furthermore, the seminal vesicle weight of the F1 generation cohort 1A animals (0.863 g) was in a similar range as the weight in cohort 1B animals (0.85 g).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS
- F1 pups on PND 4: A few pups showed spontaneous findings, such as post mortem autolysis, hematoma, diaphragmatic hernia, hydronephrosis, dilated renal pelvis and hydroureter. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or higher incidences
- F1 weanlings not selected for cohorts on PND 22: no effects

COHORTS
F1A and F1B: no effects
Histopathological findings:
no effects observed
Description (incidence and severity):
F1 PUPS
- F1 weanlings not selected for cohorts on PND 22: not conducted

COHORTS
- F1A: no effects
- F1B: not conducted
Other effects:
no effects observed
Description (incidence and severity):
F1 PUPS
- Number of pups per dam, rates of liveborn, stillborn and dead pups were evenly distributed
- Lactation index: 99%, 99%, 100%, and 99% in control, 1250, 3750, 12500 ppm
- Sex ratio not affected

COHORTS
- Differential ovarian follicle count in F1A revealed no significant differences
- F1A and F1B: no effects on estrous cycle
- F1A: no effects on sperm measures
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: developmental toxicity no adverse effects
Remarks on result:
other:
Remarks:
about 1221 mg/kg bw/d in males and 1521 mg/kg bw/d in females),
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general systemic toxicity no adverse effects
Remarks on result:
other:
Remarks:
about 1221 mg/kg bw/d in males and 1521 mg/kg bw/d in females),
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1B)
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general systemic toxicity no adverse effects
Remarks on result:
other:
Remarks:
about 1221 mg/kg bw/d in males and 1521 mg/kg bw/d in females),
Critical effects observed:
no
Reproductive effects observed:
no

Intake of test substance Cohort F1A

 

 

Test group 11

(1000 ppm)

Test group 12

(4000 ppm)

Test group 13

(12500 ppm)

Males

154.4 (102.5 / 276.5)

464.2 (319.9 / 840.0)

1500.9 (965.8 / 2575.1)

Females

186.2 (159.0 / 285.0)

580.7 (505.0 / 865.2)

1668.8 (1305.4 / 2563.2)

 

Intake of the test substance Cohort F1B

 

 

Test group 11

(1250 ppm)

Test group 12

(3750 ppm)

Test group 13

(12500 ppm)

Males

161.3 (115.4 / 286.1)

495.4 (352.8 / 881.1)

1415.6 (952.8 / 2473.4)

Females

184.4 (157.0 / 282.5)

583.2 (496.9 / 870.0)

1676.2 (1385.8 / 2516.7)

 

Intake of test substance F0 parental animals

 

 

Test group 01 (1250/625 ppm)

Test group 02 (3750/1875 ppm)

Test group 03 (12500/6250 ppm)

F0 males (premating)

F0 males (mating and postmating)

126.3 (86.4 / 144.6)

80.8 (69.9 / 100.1)

407.1 (264.1 /440.0)

249.6 (227.4 /296.0)

1187.5 (778.3 / 1338.7)

778.7 (711.3 / 911.6)

F0 females (premating)

156.8 (131.2 / 223.1)

564.9 (455.8 / 826.6)

1571.0 (1295.0 / 2106.8)

F0 females

- gestation period

- lactation period

 

127.0 (100.9 / 161.9)

138.7 (86.4 / 207.0)

 

444.7 (367.3 /531.2)

427.9 (258.1 /677.3)

 

1282.7 (1021.0 / 1582.3)

1404.2 (893.6 / 2031.5)

 

Conclusions:
Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 3750 ppm (about 405 mg/kg bw/d) in the F0 parental males, based on decreased body weight/body weight gain at 12500 ppm. In the F0 parental females as well as adolescent and adult F1 offspring this NOAEL is 12500 ppm (about 1221 mg/kg bw/d in males and 1521 mg/kg bw/d in females). The NOAEL for fertility and reproductive performance for the parental rats is 12500 ppm (about 1221 mg/kg bw/d in males and 1521 mg/kg bw/d in females), the highest tested dose. The NOAEL for developmental toxicity in the F1 progeny is 12500 ppm (about 1221 mg/kg bw/d in males and 1521 mg/kg bw/d in females).
Executive summary:

The test substance was administered to groups of 25 male and 25 female healthy young Wistar rats for test groups 00 - 03 as a solution in drinking water of different concentrations (0, 1250, 3750 and 12500 ppm). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts which were subjected to specific postweaning examinations. The study was terminated by the terminal sacrifice of the F1 rearing animals of cohort 1B. Test drinking water containing the test substance was offered continuously throughout the study.

The following test substance-related adverse effects/findings were noted:

12500 ppm

F0 PARENTAL ANIMALS (11R278M0/F0/L1)

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY

• Decreased body weights in the males from premating day 28 onwards until the end of the study, average final inlife weight 7% below control, decreased body weight gain during pre-mating (9% below control)

F1 PUPS (11R278L1)

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A (11R2781A)

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1B (11R2781B)

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

3750 ppm

F0 PARENTAL ANIMALS (11R278M0/F0/L1)

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/

PATHOLOGY

• No test substance-related adverse findings

F1 PUPS (11R278L1)

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A (11R2781A)

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1B (11R2781B)

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

1250 ppm

F0 PARENTAL ANIMALS (11R278M0/F0/L1)

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/

PATHOLOGY

• No test substance-related adverse findings

F1 PUPS (11R278L1)

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A (11R2781A)

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1B (11R2781B)

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

The NOAEL (general toxicity) was considered to be 3750 ppm, NOAEL (fertility and reproduction) was 12500 ppm and the NOAEL (developmental) was 12500 ppm.

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
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: other: = about 785 mg/kg bw/d for males and 1273 mg/kg bw/d for females
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
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

To investigate the potential of the test substance to cause adverse effects on the reproductive system of rodents, a modified extended one-generation study according to OECD 443 and in compliance with GLP regulations was performed (BASF 2019). The test substance was administered continuously to groups of 25 male and female Wistar rats in drinking water of different concentrations (0, 1250, 3750 and 12500 ppm). Pups of the F1 litter were selected and assigned to 2 different cohorts. There were no test substance-related mortalities, adverse clinical observations or effects on food consumption in any of the groups. Body weights and body weight change of low and mid-dose F0 males and females, as well as all male and female F1 rats, were not affected by treatment.The body weights of the high-dose F0 males were below the concurrent control values from premating day 28 onwards until the end of the study, most of the time the difference was statistically significant. The average final in-life weight of these males was 7% below control. Accordingly, body weight gain was decreased compared to control during premating days 0 - 7 and 0 - 70 (about 8% and 9%, respectively). It was suggested that it was treatment-related and was considered as a sign of an adverse systemic effect. Regarding clinical pathology, pathology, organ weights, macroscopy, microscopy, and differential ovarian follicle count no treatment-related adverse findings were noted. No effects on fertility, reproductive performance, and developmental toxicity were reported.A statistically significant delay in vaginal opening of about one day beyond the concurrent control was observed in the female F1 offspring of the high-dose group (12500 ppm). The delay is also slightly (less than a day) beyond the historical control range of the test facility. However, pubertal age in test groups 01 (1250 ppm) and 02 (3750 ppm) as well as in concurrent control were all above the upper limit of the historical range, while the statistically significantly higher value in test group 03 (12500 ppm) was again only slightly above concurrent control. This apparent delay was solely due to 4 high-dose individuals entering puberty after the age of 36 days. This and the still rather small average difference to the control of about one day indicates that the later onset of puberty in test group 03 is not a specific effect on the timing of puberty. In addition, none of the other endocrine-sensitive parameters like anogenital distance, or estrous cyclicity in the F1A and B offspring brought beyond puberty, or the integrity of sexual organs in these females including differential ovarian follicle count, indicated any effect of the test item. Thus, the apparently later entry of test group 03 into female puberty is likely to be incidental.Thus, the NOAEL for general, systemic toxicity is 3750 ppm (about 405 mg/kg bw/d) and 12500 ppm (about 1221 mg/kg bw/d in males and 1521 mg/kg bw/d in females) in F0 parental males and in F0 parental females as well as adolescent and adult F1 offspring, respectively. The NOAEL for fertility and reproductive performance for parental rats and developmental toxicity for F1 progeny is 12500 ppm, the highest tested dose.

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.

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 analogues. 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

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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.

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Remarks:
BASF AG, Department of Toxicology
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH facilities, 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: 24 - 29 weeks
- Weight at study initiation: ca 2.7 kg
- Housing: singly in wire cages (type K 300/8, EBECO, Becker & Co., Castrop-Rauxel, FRG)
- Diet (e.g. ad libitum): KLIBA rabbit laboratory diet 24-341-4 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
- 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: artificial insemination
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 7 - 19 of gestation
Frequency of treatment:
6 hours/day
Duration of test:
29 days
Remarks:
Doses / Concentrations:
0.5, 2.5 and 10 mg/l
Basis:
nominal conc.
target concentration
Remarks:
Doses / Concentrations:
0.51 ± 0.014, 2.51 ± 0.150 and 9.8 ± 0.66 mg/l
Basis:
analytical conc.
gas chromatograph monitoring
No. of animals per sex per dose:
15 (in triplicate of 5 animals per 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 difference between the body weight on the day of weighing and the body weight on the day of the previons weighing was calculated as a group mean. The same was done for the 3 different study priods:
* preflow period (day 0- 7 p.i.)
* exposure period (days 7 - 19 p.i.)
* observation period (days 19 - 29 p.i.).
These values are defined as body weight change. Moreover, the corrected body weight gain (body weight on day 29 p.i. minus the body weight on day 7 p.i. 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 29 p.i. the dams were sacrificed by intravenous injection of a pentobarbital and the fetuses removed by cesarean section.

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. 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, preand 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:
Maternal toxicity: Slight, statistically significant retardation in body weight gain of the dams of the 10 mg/l group in the exposure period. Indications of irritating effect on dam's eyes during exposure period.
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 29 p.i.) 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 one female (test group 1) was excluded from the above mentioned calculations since she did not conceive.

 

*Clinical signs and findings: there were no substance-induced clinical signs or findings in test groups 0, 1 and 2 at any time of the study period (preflow, exposure, post-exposure observation). The animals of test group 3 showed reddish eyes, eyelid closure and slight, aqueous discharge from eyes after exposure. Towards the end of exposure, these symptoms were also observed before exposure. The animals were free from abnormal clinical signs and findings during preflow and post-exposure observation. One animal showed reduced defecation at the start of the post-exposure observation period.

 

- Body weight:

The body weights of all test groups (1 - 3) compared to the control (0) were not statistically significantly influenced.

 

- Body weight change:

The body weight change of test groups 1 - 2 compared to the control (0) was not statistically significantly influenced. In test group 3 there was a statistically significant retardation of the body weight (p < 0.05) between day 7 - 10 p.i. compared to the control. Over the total exposure period (days 7- 19 p.i.) a slight retardation in body weight increase can be observed. Both findings are indications of a substance related effect, which is more pronounced in the first phase of the exposure period. This is confirmed by a statistically significant higher increase of the body weight in the post-exposure observation period, which can be interpreted as a recovery after end of exposure.

Corrected body weight gain (net maternal body weight change): the results of the corrected body weight gain (body weight on day 29 p.i. minus body weight on day 7 p.i. minus weight of the uterus before it was opened) do not show clearly dose-related differences between the groups.

 

- Examinations of the dams at termination:

*Necropsy findings: all of the findings which were recorded for the does are spontaneous ones (i.e. inflammation in fatty tissue, abscess in kidney, blind ending uterine horn(s)), and are not related to the exposure of the does.

*Uterus weight: the uterus weight of the animals of the substance-treated groups do not show any significant differences in comparison to the controls. The observable differences are in the range of biological variation.

*Reproduction data of dams: the conception rate varied between 93 and 100%. Concerning all groups, there were no substance-related and/or statistically significant differences in the conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation loss, the number of resorptions or viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

 

2) Examinations of the fetuses:

 - Sex distribution of fetuses:

The sex distribution in test groups 1-3 (0.5 - 10 mg/l) was comparable with the control group. The observable differences are without any biological relevance.

- Weight of placentae:

The mean placental weights in test groups 1, 2 and 3 (0.5, 2.5 and 10 mg/l) were not influenced by the administration of the test substance to the does. The differences observed in comparison to the control are without any dose-response relationship and without any biological relevance. The marginally reduced mean placental weights in the substance-exposed groups are not related to the test substance exposure, but are caused by the incidentally increased number of live fetuses/doe in these groups in comparison to the controls.

- External examination of the fetuses:

The external examination of the fetuses revealed no malformations and only one type of variation (pseudoankylosis) in one fetus each of test groups 1 and 2 (0.5 and 2.5 mg/l) and 2 fetuses of test group 3 (10 mg/l). This common finding is also present at a comparable incidence in the historical control.

- Soft tissue examinations of the fetuses:

The examination of the organs of the fetuses revealed no malformations in any group.

- Skeletal examination of the fetuses:

Various malformations of the sternum and/or the vertebral column were seen in 3 fetuses of the control and the 0.5 mg/l group and in 5 fetuses of the 2.5 and 10 mg/l group. No other skeletal malformations were recorded for any group.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
A read-across justification is provided in the IUCLID Chapter "Assessment reports"
Principles of method if other than guideline:
Developmental study, where rats were exposed on gestation days 1-19
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI, USA
- Weight at study initiation: 200 - 300 g
- Housing: in standard metal cages equipped with automatic water dispensers
- Diet (e.g. ad libitum): NIH-07 lab chow (Ziegler Bros., Garden, MA); ad libitum
- Water (e.g. ad libitum): tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 60
- 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: Hinners-type chamber
- Method of holding animals in test chamber: in stainless steel wire mesh cages within the exposure chambers
- System of generating particulates/aerosols: A constant flow of test substance was mixed with a known volume of heated compressed air, resulting in instantaneous vaporization of the alcohol. This vapor-air mixture was introduced into the mainstream of the chamber airflow upstream from an orifice, and the resulting turbulence produced uniform mixing of the test chemical throughout the exposure chamber
- Temperature, humidity, pressure in air chamber: temperature of 25 ± 2°C; relative humidity of 50 ± 15%
- Air flow rate: ca. 0.5 m3/minute


TEST ATMOSPHERE
- Brief description of analytical method used: using a Miran 1A infrared-Analyzer at every hour
- Samples taken from breathing zone: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Charcoal tube samples were drawn 2 days/week and analyzed by gas chromatography with partial verification of these methods using spiked samples of known concentration.
Means from the continuous monitoring were equal to the target value of 14 mg/L, with standard deviations not exceeding 5% of the means. Concentrations from the secondary monitoring method (gas chromatograph analysis of samples captured on charcoal tubes) were frequently 10-20% lower than the means from the primary method. However, results from spiked samples were often similarly below the concentrations supplied. Consequently, the figures from the primary monitoring method (infrared analyzer) are cited.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 1 - 19 of gestation
Frequency of treatment:
7 h/day
Duration of test:
20 d
Remarks:
Doses / Concentrations:
14 mg/L
Basis:
nominal conc.
highest concentration technically achievable
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data



DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: weighed daily for the first week and weekly thereafter


FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No


WATER CONSUMPTION: Yes
- Time schedule for examinations: on gestation days 7, 14, 20


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus with ovaries
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- 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: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
Data were analyzed using multivariate analysis of variance (MANOVA) and analysis of variance (ANOVA); p< 0.05 was accepted as statistically significant. Three separate analyses were performed using exposure group as the independent variable. For the litter data (numbers of corpora lutea, resorptions, females per litter and males per litter, and weight of females per litter and of males per litter), a one-way ANOVA/ANOVA design was used. If a significant MANOVA was observed, individual ANOVAs were performed, and if these were significant, Bonferroni corrections were made comparing the individual exposure group with controls. A second analysis, for the weight data, used a litter per exposure group X day ANOVA. A third analysis, for the feed and water consumption data, used a litter per exposure group X week MANOVA/ANOVA design. For the ANOVAs, the probabilities of all within-litter main effects and interactions were corrected with the Greenhouse-Geisser estimate of Box's epsilon.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Comparing to the control group maternal body weights were decreased slightly, but this difference was not statistically significant. Food consumption was decreased, but water consumption unchanged.
Dose descriptor:
NOAEC
Effect level:
14 mg/L air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Number of corpea lutea, resorptions, gender ratio and fetal weights were not affected by treatment.
Small, but nonsignificant, reversible delays in ossification of caudal vertebrae, sternum, metacarpals and hindpaw phalanges were reported.
No malformations were observed.
Dose descriptor:
NOAEC
Effect level:
>= 14 mg/L air
Basis for effect level:
other: no adverse effects observed
Abnormalities:
not specified
Developmental effects observed:
not specified

Results:

1-Pentanol Control
Mean maternal weight (g)
day 0 260 ± 22 243 ± 25
day 7 297 ± 21 262 ± 24
day 14 291 ± 23 291 ± 26
day 20 348 ± 25 354 ± 32
overall gain 88 111
Mean feed consumption (g)
week 1 92 ± 14 108 ± 14
week 2 110 ± 10 124 ± 12
week 3 99 ± 12 118 ± 10
overall mean 100 ± 14 117 ± 13
Mean water intake (g)
week 1 222 ± 33 204 ± 46
week 2 223 ± 43 276 ± 93
week 3 288 ± 45 265 ± 123
overall mean 244 ± 51 248 ± 96
Mean corpora lutea/litter 13 ± 3 14 ± 4
Mean resorptions/litter 0.2 0.4
Mean no. females /litter 7 ± 1 8 ± 2
Mean no. males /litter 5 ± 2 7 ± 2
Mean fetal weight (g)
males 3.11 ± 0.31 3.19 ± 0.2
females 3.32 ± 0.26 3.28 ± 0.27
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.