Linalool

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 March - 08 October 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study was conducted according to OECD 421 and under GLP conditions. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. Linalool and Dehydrolinalool have almost identical chemical structures. The only difference is the triple bond at position 1 in Dehydrolinalool compared to a double bond at the same position in Linalool. Both substances have almost identical physical-chemical properties. Therefore, it is assumed that toxicological properties are as well comparable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Preliminary Reproduction Toxicity Screening Test (Precursor Protocol of GL 421)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethyloct-6-en-1-yn-3-ol

EC Number:

249-482-6

EC Name:

3,7-dimethyloct-6-en-1-yn-3-ol

Cas Number:

29171-20-8

Molecular formula:

C10H16O

IUPAC Name:

3,7-dimethyloct-6-en-1-yn-3-ol

Details on test material:

- Name of test material (as cited in study report): dehydrolinalool
- Molecular formula: C10H16O
- Molecular weight: 152.23
- SMILES Code: CC(=CCCC(C)(C#C)O)C
- InChI : 1/C10H16O/c1-5-10(4,11)8-6-7-9(2)3/h1,7,11H,6,8H2,2-4H3
- Physical state: liquid
- Stability under test conditions: Stable for 3 weeks in vehicle in refrigerator
- Storage condition of test material: In rape seed oil in refrigerator

Test animals

Species:

rat

Strain:

other: Fü-Albino (RORO)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Biological Research Laboratories Ltd., CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: no data
- Weight at study initiation: (P) Males: 266-272 g; Females: 180-184 g
- Housing: Individually (female + male during mating), wire mesh cages until mating, macrolon cages during post-mating period
- Diet: Kliba 343 pellets
- Water: Ad libitum, tap water
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): fully air conditioned, no further details
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not relevant

Vehicle:

other: rape seed oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test compound formulated in vehicle and stored in a refrigerator. During dosing the solution was moved by the use of a magnetostirrer. Preparation is done each 3 weeks. Formulation in rape seed oil is homogenous and stable for 3 weeks.

DIET PREPARATION
- Administration not by diet (gavage)

VEHICLE
- Amount of vehicle: 5 mL/kg bw/day for each dose

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Overnight
- Proof of pregnancy: Copulary plug, referred to as day 0 of gestation
- Further matings after two unsuccessful attempts: Yes (for 13 consecutive days with same male)
- After successful mating each pregnant female was caged: Individually
- Any other deviations from standard protocol: females without evidence of coitus were autopsied after the mating period and their organs grossly examined. Their uteri were examined for implantation sites. Some animals without evidence of coitus were found to be pregnant and have fetuses at autopsy. The fetuses were discarded and judged as resorptions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Not relevant

Duration of treatment / exposure:

Males: 2 weeks prior to mating and during 2 week mating period up to day 4 of lactation of the respective litter
Females: 2 weeks prior to mating and during 2 week mating period, during gestation and lactation up to day 4 of lactation

Frequency of treatment:

Daily

Details on study schedule:

No data

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A preceding dose-range finding study on 8 mated female rats per group was done. They were treated with dehydrolinalool at doses of 0, 250, 500 or 1000 mg/kg/day. The highest dose showed significant clinical observations (piloerection, reduced activity, hypersalivation) so it was reduced to 750 mg/kg/day. Clinical observations disappeared after this reduction and therefore 750 mg/kg/day was selected as highest dose. Oral route was selected because it is the recommended administration route by regulative authorities.
- Rationale for animal assignment: Random

Positive control:

Not necessary

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations: All changes of behaviour and general condition, signs of pharmacological effects etc.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Weekly
Females: Weekly during premating and mating, on day 0 of gestation, weekly thereafter and on days 1 and 4 of lactation (birth date of F1-generation = day 1 of lactation)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable (gavage study)

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: Testes weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No, all offspring sacrificed on day 4

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 abnormalities

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 were sacrificed and autopsied after day 4 of lactation of respective female
- Maternal animals: All surviving animals were allowed to litter spontaneously and to rear their young up to day 4 of lactation. Date of parturition was recorded (birth date = day 1 of lactation). Then the animals were killed. Females that did not litter were sacrificed about one week after expected date of parturition. Females which died during study were autopsied.

GROSS NECROPSY
- Gross necropsy consisted of macroscopical examination of kidneys, lungs and liver. Testes was weighed. Uteri was examined for implantation sites. Ovaries were preserved and the number of corpora lutea was recorded.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic examination) as follows: Externally examined for anomalies.

GROSS NECROPSY
- Gross necropsy consisted of visceral examination (anomalies) according to the method of Barrow and Taylor. Visceral examination in the 50 and 200 mg/kg/day groups was limited to urogenital system. Determination of sex was done at necropsy.

Statistics:

Descriptive statistics + significance tests (chi-square and fisher's exact test for categorical values, ANOVA and Dunnett's test for normally distributed values, Kruskal-Wallis-Test and Mann-Whitney-Wilcoxon-Test for all other parameters).

Reproductive indices:

Female mating index, female fertility index, female gestation index, male mating index, male fertility index.

Offspring viability indices:

Pup live birth index, pup viability index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Hypersalivation in both sexes at high dose group, sedation/ataxia in some females of high dose group. Hypersalivation was not considered an adverse effect. 3 females of the 750 mg/kg bw/day group died/were sacrificed during pre-mating period, another 3 died during delivery.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
At 750 mg/kg the pup live birth index and pup viability index were slightly reduced, when compared with the concurrent control. This is likely related to observed maternal toxicity. Moreover, dams with delivery complications were observed at the dose of 50 mg/kg (1 dam) and at 750 mg/kg (3 dams). The delivery complications may be related to sedating effect of the test substance as evidenced by clinical signs at high dose level (750 mg/kg bw/day).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Pup viability index significantly reduced in high dose group and possibly by toxic effect in treated dams.

GROSS PATHOLOGY (OFFSPRING)
Most frequently observed abnormalities in 750 mg/kg/day group (hydronephrosis, convoluted and/or hydroureter and retardation in renal and testicular development) were mainly noted in pups; belonging to mothers with delivery problems. They are likely to be linked to maternal toxicity.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

viability

other: Abnormalities of urinary system in 750 mg/kg/day group

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

other: Abnormalities of urinary system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Any other information on results incl. tables

Linalool and Dehydrolinalool are liquids at room temperature and have a boiling point of 197 - 198°C. The slight differences in vapor pressure (Linalool: 0.27 hPa at 25°C, Dehydrolinalool: 0.11 hPa at 20°C), water solubility (Linalool: 1.56 g/L at 25°C, Dehydrolinalool: 2.45 g/L at 20°C), and log Pow (Linalool: 2.9 at 20°C, Dehydrolinalool: 2.61 at 25°C) are mainly the result of different testing temperatures. Vapor pressure favors evaporation, water solubility and logPow indicate that both substances may favor organic apolar environment.

 

Acute oral and dermal toxicity data reveal for both substances an LD50 of more than 2000 mg/kg bw. Acute inhalation toxicity was not tested by standard means. However, there were no deaths at a concentration of 3.2 mg Linalool/L and 1.0 mg Dehydrolinalool/L. Thus, the LC50s are greater than 3.2 mg Linalool/L and 1.0 mg Dehydrolinalool/L. Linalool and Dehydrolinalool are both skin and eye irritants as pure substances. Dilutions of 30% concentration and below for both substances show no eye irritant potential. Neither Linalool nor Dehydrolinalool showed sensitizing potential in human studies. It was shown that oxidation of Linalool should be prevented due to strong skin sensitising properties of the decomposition products.Therefore, Linalool is usually protected by antioxidants.

Overall, mutagenicity and genotoxicity testing was unremarkable. Both substances were negative in the Ames test and in an in vivo micronucleus test. Linalool was also negative in an in vitro chromosome aberration test and in an in vitro forward mutation testing. Although Dehydrolinalool was positive in the in vitro chromosomal aberration test in the absence of metabolic activation, the next higher Tier test i.e. the in vivo MNT in the bone marrow was clearly negative. 

Two repeated toxicity studies, 2 reprotoxicity screening studies, and a developmental toxicity study using gavage application for Linalool (either applied in coriander oil at 72.9% Linalool or as pure substance) and / or Dehydrolinalool showed the following consistent results: Histopathologically, male animals had alpha-2u-globulin nephropathy after application of both substances. Liver (and kidney) weights were increased in both sexes with some indication of metabolic enzyme induction in the liver. Hypersalivation and sedation / ataxia were consistently noted in almost all studies. In one oral study, gavage application resulted in (fore)stomach lesions which might be the result of the irritant properties. In a repeated dermal toxicity study, the skin irritant properties of Linalool were confirmed. The relevant oral NOAEL for Linalool was determined to be 117 mg/kg bw/d; Dehydrolinalool has an oral NOAEL of 200 mg/kg bw/d. The difference in the NOAELs is the result of the dose-setting regime. The NOAEL for dermal DNEL was 250 mg/kg bw/d.

Linalool (applied in coriander oil at 72.9% Linalool) was only tested for female fertility without showing an effect. Dehydrolinalool, however, was tested for male and female fertility and showed no adverse effect. Linalool was tested with regard to developmental toxicity / teratogenicity in two studies: screening test (applied in coriander oil at 72.9%) and full guideline compliant developmental toxicity study (pure substance). Both studies showed no evidence of adverse effects on foetuses and pups at doses which were not maternal toxic. Dehydrolinalool was also tested negative for this endpoint in a screening assay. From the data it is concluded that both substances are neither developmental toxic nor teratogenic at doses not being maternally toxic. However, at maternal toxic doses, reduced litter sizes and increased pup mortality was noted for both substances.

 

Applicant's summary and conclusion

Conclusions:

Several and significant effects of dehydrolinalool were seen in females (sedation/ataxia) and offspring (abnormalities of the urinary system, reduced pup live birth index and pup viability) at the highest dose group of 750 mg/kg/day. For males, no effects were seen up to the highest dose level. The NOAEL for females and offspring was set at 200 mg/kg/day, for males the NOAEL was established as 750 mg/kg bw/day.

Executive summary:

Dehydrolinalool was tested in a preliminary reproductive toxicity screening test (OECD) in rats. 20 animals per sex and group were given the test article formulated in rape seed oil at doses of 0 (control), 50, 200 or 750 mg/kg/day (administration volume 5 mL/kg/day). The administration period for both sexes was through premating (2 weeks), mating (up to 2 weeks) and gestation up to day 4 of lactation.

 

3 females died/were sacrificed during pre-mating period and 3 females died/were sacrificed with delivery complications at the highest dose group. Body weight development and food consumption was comparable in all experimental groups. Hypersalivation was observed in males and females at 750 mg/kg throughout the study. However, this was not considered relevant for NOAEL derivation. Females at the highest dose groups showed sedation/ataxia during gestation and lactation. The duration of gestation, mean number of implantations, resorption rate, mean number of pups per litter, mean pup weight, sex ratio were not significantly affected by the treatment in either dose group. In addition, no signs of teratogenic action were observed in any pup of dams which survived the scheduled date of necrospsy in any dose group.

However, at 750 mg/kg the pup live birth index and pup viability index was slightly reduced, when compared with the concurrent control. Moreover, dams with delivery complications were observed at the dose of 50 mg/kg (1 dam) and at 750 mg/kg (3 dams). No delivery complications were noted in the mid dose group (200 mg/kg) and therefore the finding at low dose level was not considered adverse due to missing dose-response relationship. The incidence of abnormalities in the urinary system and of renal and testicular development was increased at 750 mg/kg mainly due to pups of dams which died or were sacrificed during the delivery. Reduced live birth index is as well to a great extent realted to delivery problems.

 

It may be concluded that dehydrolinalool does not adversely affect mating and fertility of males and females when administered in rape seed oil by oral gavage to rats from 2 weeks premating through day 4 of lactation up to 750 mg/kg/day, the highest dose tested. Delivery complications were noted at 50 and 750 mg/kg/day. The pup live birth index and pup viability index was slightly decreased at the maternally toxic dose of 750 mg/kg/day. An increased incidence of abnormalities of the urinary system and of impaired renal and testicular development was noted in pups of mothers treated with 750 mg/kg/day. However, most of the observed abnormal pups belong to those dams which died or were sacrificed moribund during delivery. No adverse effects on the reproductive indices and on the developing conceptus/pup were noted up to 200 mg/kg/day. The no-observed-adverse-effect-levels (NOAEL) were set at 200 mg/kg/day for the offspring and dams. The NOAEL was 750 mg/kg bw/day for males.