Citronellal

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
See read across justification document

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read across justification document

3. ANALOGUE APPROACH JUSTIFICATION
See read across justification document

4. DATA MATRIX
See read across justification document

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed according to Guideline 421 with acceptable restrictions: only summary report available; study acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

(no gross examination of pups)

GLP compliance:

yes

Species:

rat

Strain:

other: Sprague-Dawley Crj:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan, Inc.
- Age at study initiation: (P) 10 wks

- Fasting period before study:

- Housing: Metal bracket typed wire mesh floor gauge; stainless tray with floor materials for experimental animals(White flakes Charles River Laboratories Japan, Inc. ), was used for pregnant rats after Day 17 day of their pregnancy.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: Solid feed (CRF-1, Oriental Yeast Co., Ltd.); ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23+/-3
- Humidity (%): 55+/-10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/23

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The solution prepared was found to be stable for 5 hours after the preparation. Accordingly, the
solution was prepared everyday at the time of use.


VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg

Details on mating procedure:

- M/F ratio per cage: 1/1
- After successful mating each pregnant female was caged (how): 1 female rat/cage
- Length of cohabitation: up to 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
Confirmation of pregancy, if implantations were found in the uterus

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Premating exposure period: 14 d
Total exposure period: males 46 d, females from 14 d before mating up to day 3 of lactation

Frequency of treatment:

daily

Details on study schedule:

Terminal killing: males on day 47; females on day 4 of lactation
No further data available

Remarks:

Doses / Concentrations:
40, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose finding study and literature.

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: yes; daily observations

DETAILED CLINICAL OBSERVATIONS: yes; daily observations

BODY WEIGHT: Yes
- Time schedule for examinations: during 14 day premating period on days 1, 2, 5, 7, 10 and 14; males thereafter weekly and on day 46; females on day 0, 1, 3, 5, 7, 10, 14, 17 and 20 of gestation, and on lactation day 0, 1, and 4.
Body weight change was calculated; males: between Day 1 and Day 46 of administration; females: between Day 1 and Day 14 (premating), Day 0 and Day 20 (gestation), and Day 0 and Day 4 (lactation).

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: on same day as body weight determinations except for Day 0 of pregnancy and Day 0 of lactation.

Oestrous cyclicity (parental animals):

From all the females, vaginal smear samples were collected on the successive days
from Day 10 before administration to Day of successuful mating and stained with
Giemsa solution to determine their estrous cyclicity phase (Proestrus, early estrus, late
estrus, or anestrum) as well as checking for any irregularity (normal, continuing
anestrum, or irregular) under the light microscope.

Sperm parameters (parental animals):

not assessed

Litter observations:

PARAMETERS EXAMINED:
Number of pups born,
Number of life pups born,
Number of dead pups born,
Sex of pups born

All newborns were observed for its survival or death, general conditions, and appearance once a day from the last day of delivery until the day of necropsy (Day 4 of lactation). Body weight was measured on Days 0, 1 and 4 of nursing and the mean value per litter was obtained respectively for males and females.


GROSS EXAMINATION OF DEAD PUPS:
Dead pups were immediately necropsied.

Postmortem examinations (parental animals):

SACRIFICE
via exsanguination under ether anesthesia
- Male animals: All surviving animals on day 47.
- Maternal animals: All surviving animals on day 4 of lactation or Day 26 of pregnancy (if no delivery until Day 25 of pregnancy) or immediately
after finding any dead pups.
For males and females with unsuccessful copulation: on the next day after the mating period.


GROSS NECROPSY
no data given

ORGAN WEIGHTS
Absolute and relative weights of testis, epididymis, ovary (each right and left). Absolute weight was divided by the body weight on the necropsy day, and then multiplied by 100 to calculate the relative weight. For pregnant females, the number of implantations and corpus luteum of pregnancy were
counted.

HISTOPATHOLOGY
Main organs and tissues (not further specified), stomach (glandular stomach, forestomach and border), testis, epididymis and ovaries.
Samples were paraffin-embedded and stained with hematoxylin-eosin.

Postmortem examinations (offspring):

Pups were observed for external body surface (including inside the mouth), and euthanized by CO2 inhalation method on Day 4 of lactation. Organs and tissues throughout the body were macroscopically observed.

Statistics:

Bartlett assay. If the results had equal variance, one-way analysis of variance method and Dunnett method was conducted.
If the results had unequal variance, Kruskal-Wallis method and Mann–Whitney U test was performed:
Body weight, increase in body weight and increase rate of body weight, food consumption, absolute organ weight and relative organ weight,
number of corpora lutea, number of implantation sites, implantation index, total number of pups born, number of live pups born, delivery index,live birth index, sex ratio, number of dead pups born, gestation length, number of live pups on Day 4 of nursing, and viability index on day 4.

Kruskal-Wallis method and Mann–Whitney U test was performed:
Histopathology

Multisample X2 test was performed and Two sample X2 test was performed. However, when results failed the Multi-sample X2 test or Two-sample X2 test, then Fisher’s exact test was used:
Estrous cyclicity, copulation index, Fertility index, birth index and nursing index.

Reproductive indices:

Copulation index [(Number of pairs with sucessful copulation/Number pairs mated) x100]
Fertility index [(Number of pregnant females/Number of pairs with sucessful copulation) x100]
Implantation index [(Number of implantation sites in the uterus/Number of corpora lutea)x100]
Birth index [(Number of females with live pups delivered/Number of corpora lutea)x100]
Delivery index [(Number of pups born/Number of implantation sites)x100],
Live birth index [(Number of live pups born/number of pups born)x100],
Gestation index [(Number of females with live pups delivered/number of pregnant females)*100]
Nursing index [(Number of females with nursing live pups/Number of females with normal delivery)x100]
Sex ratio [Number of male pups/Number of female pups]
Gestation length [Number of days from Day 0 of pregnancy until Day 0 of nursing(last day of delivery)]

For all the mated females, delivery condition and maternal behavior were observed from Day 21 of pregnancy until the last day of delivery.

Offspring viability indices:

Viability index on day 4 [(Number of live pups on day 4/Number of live pups born) x100]

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Males showed no abnormalities in any dose group.
One of the females in the 1000 mg/kg group showed a temporal decrease in locomotor activity on Day 7 of administration. Another female showed temporal salivation on Day 9 and Day 10 of gestation.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
1000 mg/kg bw/d - males:
- body weight: from day 5 to 46 lower body weight than control group
- mean terminal body weight on day 46: 475.8 g compared to 502.5 g in controls, decrease of 5.3 % (no significant difference)
- A significantly reduced body weight gain was observed (no further data).
- food consumption: significant decrease on day 5 only, on other days comparable or higher than in control group
1000 mg/kg bw/d - females:
- body weight: comparable to control during premating period; lower body weight during gestation and lactation period
- mean terminal body weight on lactation day 4: 313.8 g compared to 325.3 g in controls, decrease of 3.7% (no significant difference)
- A significantly reduced body weight gain was observed during gestation (no further data).
- food consumption: significant decrease on day 5 of the premating period, further non-significant decrease on gestation day 10,
on other days comparable or higher than in control group
Other dose groups - both sexes:
- Body weights: comparable or higher than in the control groups
- Food consumption: Significantly lower food consumption was observed on Day 5 (premating) and Day 4 of lactation. For other timepoints, no consistent trend was observed compared to controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the administration period 1/12 females showed abnormal continuous diestrum in the 40 and 1000 mg/kg-groups; no effects in control or 200 mg/kg-group. Alternations observed are not considered to have any relation to the administration of the test substance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
1000 mg/kg bw/d:
- 2/12 females were not pregnant (not significant)
- Number of total dead pups born: 6 (not significant compared to control group, i.e. 14 dead pups born)
40 mg/kg bw/d:
- 3/12 females were not pregnant (not significant)
- Non successful copulation 1/12 females (not significant)
- implantation index significantly increased compared to control
- Number of total dead pups born: 5 (not significant compared to control group, i.e. 14 dead pups born)
Other dose groups and parameters: no significant effects compared to controls

GROSS PATHOLOGY (PARENTAL ANIMALS)
1000 mg/kg group:
2/12 males showed pyelectasia, 1/12 males showed testicular and epididymis atrophy, and 1 infertile male showed yellow-white masses in epididymis. All of which were observed unilaterally.
Partial thickening of the gastric mucosa in 10/12 females and 1/21 showed dark reddening of caudate lobe in the liver.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No significant changes of absolute or relative weights of testis, epididymis or ovaries

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Male reproductive organs:
1000 mg/kg bw/d: testis: 2/12 with tubular atrophy (grading: 1 slight, 1 severe unilateral);
epididymis: 2/12 with decreased intraductal sperm (grading: 1 slight, 1 severe), 1/12 with slight spermatic granuloma
Controls: testis: 1/12 with tubular atrophy (grading: slight);
Other dose groups: no adverse effects observed
- Findings in forestomach:
1000 mg/kg bw/d - males: 2/12 with slight squamous hyperplasia
1000 mg/kg bw/d - females: 10/12 with squamous hyperplasia (4/12 slight, 6/12 moderate effect), control 1/12 with slight effect;
2/12 with ulceration, 2/12 with neutrophil cellular infiltration and 6/12 with granulation in the lamina propria,
Other dose groups and controls: no changes
- Findings in liver:
1000 mg/kg bw/d - females: 1/1 with centrilobular necrosis (no further females examined) confirmation of necropsy findings, i.e. dark reddening of caudate lobe in the liver
All groups - males: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased body weight and food consumption, histopathology of forestomach

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: NOAEL = highest tested dose

VIABILITY (OFFSPRING)
Viability index in dosed animals was slightly higher than in controls (not significant)

BODY WEIGHT (OFFSPRING)
1000 mg/kg bw/d - both sexes: significant decrease on day 0, 1, 4 after birth compared to control (see Table 1)

GROSS PATHOLOGY (OFFSPRING)
200 mg/kg bw/day: 1 case of pyelectasia and 2 cases of ureter dilatation
40 mg/kg bw/day: 2 cases with injuries to the tail and dark reddening

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased postnatal body weight gain

Reproductive effects observed:

not specified

Table 1: Body weights of pups

Sex	Day after birth	Dose group
		0	40	200	1000
male	0	6.81 ± 0.48	6.62 ± 0.64	6.43 ± 0.82	5.88 ± 0.56 ^a
	1	7.52 ± 0.58	7.50 ± 0.71	7.08 ± 1.11	6.31 ± 0.77 ^a
	4	10.77 ± 0.80	10.88 ± 1.34	10.24 ± 1.87	8.77 ± 0.84 ^a
female	0	6.33 ± 0.61	6.34 ± 0.63	6.04 ± 0.80	5.34 ± 0.70 ^a
	1	7.09 ± 0.71	7.07 ± 0.69	6.61 ± 1.13	5.69 ± 0.97 ^a
	4	10.08 ± 0.99	10.32 ± 1.50	9.71 ± 1.81	7.98 ± 1.15 ^a

significantly different from control group: ^a P < 0.01

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Citral

EC Number:

226-394-6

EC Name:

Citral

Cas Number:

5392-40-5

Molecular formula:

C10H16O

IUPAC Name:

Reaction mass of (E)-3,7-dimethylocta-2,6-dienal and (Z)-3,7-dimethylocta-2,6-dienal

Constituent 2

Reference substance name:

(Z)-3,7-dimethylocta-2,6-dienal

EC Number:

203-379-2

EC Name:

(Z)-3,7-dimethylocta-2,6-dienal

Details on test material:

- Name of test material (as cited in study report): Citral
- Analytical purity: 98.2%
- Lot/batch No.: 62938, Kurakay, Tokyo
- Physical state: insoluble, clear, serous yellowish liquid
- Stability under test conditions: verified
- Storage condition of test material: room temperature, no direct sunlight, sealed container

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased body weight and food consumption, histopathology of forestomach

2

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: NOAEL = highest tested dose

Results: F1 generation

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: decreased postnatal body weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified