Isovaleric acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Isovaleric acid is not considered to be reprotoxic, based on a NOAEL of 600 mg/kg bw and day in an oral rat screening study (highest tested dose), and based on a NOAEC of 10 mg 3-methylbutanol/L in OECD 414 inhalation studies with rats and rabbits under GLP conditions. 3-methylbutanol is rapidly oxidised to isovaleric acid in-vivo, i.e. the result can be transferred to isovaleric acid. 

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Waiving argument: The substance is registered according to Annex VII data requirements therefore no data are required.

Justification for selection of Effect on fertility via oral route:
waiving argument: valid developmental toxicity information available

Justification for selection of Effect on fertility via inhalation route:
waiving argument: valid developmental toxicity information available

Justification for selection of Effect on fertility via dermal route:
waiving argument: valid developmental toxicity information available

Effects on developmental toxicity

Description of key information

3-methylbutanol, which may be used as surrogate because it is rapidly oxidised to isovaleric acid in-vivo, caused no developmental toxicity in GLP OECD 414 guideline inhalation studies using rats and rabbits. The NOAEL for developmental toxicity was 10 mg/L in both species.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Screening study. Similar to GLP guideline study, but only one dose level tested and using low animal number.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

screening; only one dose level tested.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-15 weeks
- Weight at study initiation: 166 g (report, table IA-005)
- Fasting period before study: no data
- Housing: singly in Makrolon cages type M III
- Diet: Kliba maintenance diet mouse/rat "GLP", ad libitum):
- Water: ad libitum
- Acclimation period: from day 0 p.c. (day of supply) to day 6 p.c. (beginng of treatment)

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

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Isovaleric acid was topped up with olive oil in a volumentric flask and shaken to give the desired concentration of 150 mg/mL.

VEHICLE
- Justification for use and choice of vehicle (if other than water): poor water solubility of the test substance
- Concentration in vehicle: 150 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were carried out by BASF AG; methods and results contained in a separate report

Details on mating procedure:

- Time-mated females rats were used

Duration of treatment / exposure:

Gestation day 6 through 19

Frequency of treatment:

one per day

Duration of test:

dams were sacrificed on gestation day 20

Remarks:

Doses / Concentrations:
600 mg/kg bw and day
Basis:
actual ingested

No. of animals per sex per dose:

10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on existing information

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked in table IA-001 and -002 were included.

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: if clinical signs require examination

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 1, 3, 6, 8, 10, 13, 15, 17, 19, and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross pathological examnation; weight of unopened uterus; number of corpora lutea, implantations, resorptions, site of implantation in uterus; lungs (histopathology)

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
- Other: site of implantation

Fetal examinations:

- Weighing: Yes: all per litter
- Sex determiantion: Yes: all per litter
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes:half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

DUNNETT's test (food consumption, body weight data including fetuses, live fetuses, corpora lutea, uetrus data); FISHER's exact test (number of pregnant animals at the end of the study, mortality rate of dams, number of litters with foetal findings); WILCOXON test (proportion of fetuses with findings per litter); KRUSKAL-WALLIS and WILCOXON test (pathology; weight parameters))

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
no effects except salivation in all dams after treatment

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Isovaleric acid: Summary of reproduction data, dams (report, tables IA-010 – IA-012)

		Olive oil	Isovaleric acid
Pregnant at terminal sacrifice, N		9	9
Resorptions (mean %)	total	2.6	0.6
	early	1.4	0.6
	late	0.1	0.0
Dead fetuses, N		2	1
Live fetuses (mean %)	Total, N	69	81
	total	94.6	92.9
	females	46.6	40.2
	males	48.0	52.8
Per cent live females		49.3	43.2
Per cent live males		50.7	56.8

 

Isovaleric acid: Summary of reproduction data, fetuses (report, table IB-001)

		Olive oil	Isovaleric acid
Mean placental weights of all viable fetuses (g) (N)		0.46 (9)	0.46 (9)
Mean foetal weights (g)	total	3.6	3.5
	male	3.7	3.6
	female	3.4	3.4

 

Isovaleric acid: Summary of all foetal external, soft tissue, and skeletal observations (table IB-025)

		Olive oil	Isovaleric acid
Litters evaluated, N	N	9	9
Fetuses evaluated, N	total	71	82
	Live	69	81
	dead	2	1
Total malformations (means, %)	Foetal incidence, %	0.0	0.0
	Litter incidence, %	0	0
	Affected fetuses per litter, %	0.0	0.0
Total variations (means, %)	Foetal incidence, %	56	57
	Litter incidence, %	100	100
	Affected fetuses per litter, %	57.1	57.7

Conclusions:

Isovaleric acid was not reprotoxic (development, teratogenicity) in a rat screening study. The NOAEL for both maternal and developmental toxicity was 600 mg/kg bw, the only tested dose.

Executive summary:

The developmental toxicity of isovaleric acid (and related substances, see below) was determined in a GLP screening study that basically followed OECD 414, but using less animals (10 time-mated control and treated rats) and only one concentration (600 mg isovaleric acid/kg bw; in olive oil, oral gavage, gestation days 6-19). Dams were sacrificed on GD 20; examinations included maternal body weights, reproduction data, and pathology. Fetuses were sexed and weighed, and examined for external, soft tissue and skeletal malformations and variations. 

There were no signs of maternal toxicity nor effects on prenatal development, hence the NOAEL was 600 mg/kg bw and day for maternal toxicity, developmental toxicity, and teratogenicity under the conditions of this study (BASF, 2008).

This study is considered to be a valid screening study. It could be noted that closely related acids were also included in this study, using the same method:

	Dose (mg/kg bw and day)	NOAEL maternal toxicity (mg/kg bw and day)	NOAEL prenatal development (mg/kg bw and day)
Valeric acid	600	600	600
Isovaleric acid	600	600	600
2-methylvaleric acid	1000	1000	1000
3,5,5-trimethylhexanoic acid	300	< 300	300

 

Local irritation of the larynx and the upper and lower respiratory tract was seen with all tested acids. The effects were more significant –although still not severe – with valeric and isovaleric acid, and minimal with 3,5,5-trimethylhexanoic acid.

The above results provide evidence that the NOAEL of isovaleric acid is above 600 mg/kg bw and day in the rat following oral gavage.

Reference 2

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study Readacross is justified due to the toxicokinetic arguments described within the Endpoint Summary.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

- 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: approx. 11 weeks
- Weight at study initiation: approx. 216 g
- Housing: singly in wire cages
- Diet: KLIBA rat/mouse laboratory diet 24-343-4 10 mm pellets, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; during the exposure-free observation period ad libitum
- Water: tap water ad libitum; 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:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: the animals were exposed singly in wire cages in the whole-body inhalation chambers (volumes ca. 1100 L (test group 1, 2 and 3), and ca. 1600 L (test group 0 and parts of the test groups during the pre-flow period).
- Method of holding animals in test chamber: animals were expose din the wire cages
- Source and rate of air: the test substance was supplied by means of two continuously driven metering pumps to a vaporizer heated with a circulating thermostat. The evaporation temperature was 50-70°C. A measured stream of fresh air took up the vapours and was mixed with another stream of fresh air. After passing through a mixing device, this mixture of vapours and air was supplied to the exposure chamber.
- Temperature, humidity, pressure in air chamber: temperature and pressure in the inhalation chamber was measured continuously (generally 3 times/exposure). The relative humidity was measured at least daily.
- Air flow rate: all air flows, supply air and exhaust air were adjusted by means of flowmeters for all test groups and recorded, as a rule, 6 times/exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the test groups were analyzed by gas chromatography after absorption of 3-methylbutanol in 2-propanol. The gas chromatograph was calibrated with the test substance.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography

Details on mating procedure:

- Impregnation procedure:cohoused
- If cohoused:
- M/F ratio per cage: 1:4
- Length of cohabitation: over night
- 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:

Gestation days 6 - 15; 6 hours/day

Frequency of treatment:

daily

Duration of test:

20 days

Remarks:

Doses / Concentrations:
0.5, 2.5 and 10 mg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0.51±0.02, 2.50±0.17,and 9.8±0.66 mg/L
Basis:
analytical conc.
GC

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a pilot study. Doses up to 5 mg/L caused no toxicity in females.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0, 3, 6, 9, 12, 15, 18, and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20.
- Organs examined: uterus, ovaries, placenta

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:

Statistics:

DUNNETT's test, Fisher's exact test

Indices:

The folowing indices were calculated:
- conception rate ( %;): (number of pregnant animals/ number of fertilized animals)*100
- preimplantation loss (%): (number of corpora lutea - number of implantations/number of corpora lutea)*100
- postimplantation loss (%): (number of implantations - number of live fetuses/number of implantations)*100

Historical control data:

yes

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: marginal body weight retardation at 10 mg/L

Details on maternal toxic effects:
Transient marginal body weight reduction at 10 mg/L at beginning of the study

Dose descriptor:

NOAEC

Effect level:

2.5 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEC

Effect level:

10 mg/m³ air (analytical)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: at any dose level

Dose descriptor:

NOAEC

Effect level:

10 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

In an OECD 414 study (inhalation, rats) , 3-methylbutanol caused marginal maternal toxicity but no toxicity to reproduction (development, teratogneicity).

Executive summary:

The toxicity of 3-methylbutanol to reproduction was examined in Wistar rats (25 females per dose; whole body inhalation exposure at 0, 0.5, 2.5, and 10 mg/L; gestation days 6-15, 6 hours per day) according to OECD 414 and under GLP conditions. No maternal or developmental toxicity was noted in a pre-test at 5 mg/L.

In the main study, maternal toxicity was limited to a transient marginal body weight retardation at the start of the exposure period. Reproduction parameters were not affected at any dose. There was no effect regarding viability, development and malformations noted in the fetuses. The NOAEC for maternal toxicity was 2.5 mg/L, the NOAEC for developmental toxicity and teratogenicity was 10 mg/L, the highest tested dose (Klimisch, 1990).

Since 3-methylbutanol is rapidly oxidised to isovaleric acid, this result can be transferred to isovaleric acid. In this context it could be further noted that the same result (NOAEC 2.5 mg/L for the maternal organism and 10 mg/L for the foetal organism) was obtained in a GLP OECD 414 inhalation study with white Himalayan rabbits (15 dams/group; 0.5, 2.5, and 10 mg/L; gestation days 6-19, 6 hours/day; Klimisch and Hellwig, 1990). These results are suitable for the assessment of isovaleric acid.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Species:

rat

Quality of whole database:

Good. The negative result on isovaleric acid is supported by negative results on
valeric acid, 2-methylvaleric acid, 3,5,5-trimethylvaleric acid (BASF, 2008), and on
3-methylhexanoic acid, and 2-methylpentanoic acid (Narotsky, 1994).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

11 586 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Good. The NOAEC was 10 mg/L in two GLP guideline studies using species (rat and rabbit). 10,000 mg 3-methylbutanol (MW 88.15) correspond to 11586 mg isovaleric acid (MW 102.13), i.e. the Effect level value given above corresponds to isovaleric acid.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No fertility study was located for isovaleric acid. However,Waiving according to the REACH regulation ANNEX VIII, column 2, a fertility study is not required if valid developmental toxicity information is available. This is here the case as outlined below.

Oral exposure

The developmental toxicity of isovaleric acid (and several related substances, see below) was determined in a GLP screening study that basically followed OECD 414, but using less animals (10 time-mated control and treated rats) and only one concentration (600 mg isovaleric acid/kg bw; in olive oil, oral gavage, gestation days 6-19). Dams were sacrificed on GD 20; examinations included maternal body weights, reproduction data, and pathology. Fetuses were sexed and weighed, and examined for external, soft tissue and skeletal malformations and variations.

There were no signs of maternal toxicity nor effects on prenatal development, hence the NOAEL was 600 mg/kg bw and day for maternal toxicity, developmental toxicity, and teratogenicity under the conditions of this study (BASF, 2008).

 

This study is considered to be a valid screening study. It is further noted that that closely related acids were also included in this oral gavage study, using the same method:

 

	Dose (mg/kg bw and day)	NOAEL maternal toxicity (mg/kg bw and day)	NOAEL prenatal development (mg/kg bw and day)
Valeric acid	600	600	600
Isovaleric acid	600	600	600
2-methylvaleric acid	1000	1000	1000
3,5,5-trimethylhexanoic acid	300	< 300	300

 

Local irritation of the larynx and the upper and lower respiratory tract was seen with all tested acids. The effects were more significant –although still not severe – with valeric and isovaleric acid, and minimal with 3,5,5-trimethylhexanoic acid.

 

Further support comes from the results on 3-methylhexanoic and 2-methylpentanoic acid that were obtained with rats following oral gavage in screening so-called Chernoff-Kavlock studies, using a reduced number of test animals (20 controls, 15 treated rats per dose), two dose levels, and reduced examinations (Clinical signs, body weight, and pre-implantation loss in dams. Foetal weight at birth and at day 6. Histopathology on the pups on a screening level as follows:
Dead pups: soft tissue alterations
Externally malformed pups: skeletal and/or soft tissue alterations as indicated by the gross findings
Surviving pups: skeletal alterations in 2 pups per litter (one per sex).

In total 15 linear and branched short-chain aliphatic acids were tested (butyric, pentanoic, octanoic acid; 2-ethylbutyric, 2-methylpentanoic, bromopentanoic, 2-ethoxypentanoic, 2-ethylhexanoic, 2-propylhexanoic, 2-butylhexanoic, 2-methyloctanoic, 3-methylhexanoic, and 5-methylhexanoic acid. Positive results were only obtained with 3 substances bearing a side chain with 2 or more carbons at the 2-position (2-ethylhexanoic, 2-propylhexanoic, and 2-propylpentanoic acid), whereas 2-ethybutanoic acid was negative. The two substances that are considered to be most related to isovaleric acid, i.e. 3-methylhexanoic acid (3-MH) and 2-methylpentanoic acid (2-MP), were also no developmental toxicants is this screening study. Maternal toxicity but no developmental toxicity was noted at the highest tested doses which also represent the developmental toxicity NOAEL values of 500 mg/kg bw and day (3-MH) and 250 mg/kg bw and day (2-MP), respectively. For QSAR considerations the authors concluded that a side chain at the 2-position with two or more carbons is required for developmental toxicity (Narotsky et al., 1994).

Overall,the above results provide evidence that the NOAEL of isovaleric acid is above 600 mg/kg bw and day in the rat following oral gavage.

 

Inhalation exposure

Inhalation studies on 3-methylbutanol using rats and rabbits (OECD 414, GLP) were located. The substance is rapidly oxidised to isovaleric acid in-vivo, therefore the results may be used for assessment of isovaleric acid. More specifically, the toxicity of 3-methylbutanol to reproduction in Wistar rats (25 females per dose; whole body inhalation exposure at 0, 0.5, 2.5, and 10 mg/L; gestation days 6-15, 6 hours per day) was examined according to OECD 414 and under GLP conditions. No maternal or developmental toxicity was noted in a pre-test at 5 mg/L. In the main study, maternal toxicity was limited to a transient marginal body weight retardation at the start of the exposure period. Reproduction parameters were not affected at any dose. There was no effect regarding viability, development and malformations noted in the fetuses. The NOAEC for maternal toxicity was 2.5 mg/L, the NOAEC for developmental toxicity and teratogenicity was 10 mg/L, the highest tested dose (Klimisch, 1990).

Since 3-methylbutanol is rapidly oxidised to isovaleric acid, this result can be transferred to isovaleric acid. In this context it is noteworthy that the same result (NOAEC 2.5 mg/L for the maternal organism and 10 mg/L for the foetal organism) was obtained in a GLP OECD 414 inhalation study with white Himalayan rabbits (15 dams/group; 0.5, 2.5, and 10 mg/L; gestation days 6-19, 6 hours/day; Klimisch and Hellwig, 1990).

 

These results are suitable for the assessment of isovaleric acid. Taking default values for the rat into consideration (respiratory volume, rat: 290 L/6 hours; ECHA, Guidance Document R8, Table R.8-2), the NOAEC of 10 mg/L corresponds to 2900 mg 3-methylbutanol/kg bw and day. Taking molecular weights into consideration, this corresponds to 3360 mg isovaleric acid/kg bw and day in the rat. The use of 600 mg/kg bw and day for the derivation of DNEL values is therefore considered to be very conservative.

Justification for selection of Effect on developmental toxicity: via oral route:
Screening study on isovaleric acid (and related acids)

Justification for selection of Effect on developmental toxicity: via inhalation route:
Valid GLP guideline study on 3-methylbutanol which is rapidly oxidised in-vivo to isovaleric acid.