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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Comparable to guideline study (OECD Guideline 414), study well documented and discussed in detail with the following acceptable restrictions: exposure on gestation days 6 to 15, data on maternal observations restricted to body weight gain, no data on food consumption; study acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Study on the embryofeto-toxicity of citral in the rat

Author:

Nogueira A.C.M.A., Carvalho R.R., Souza C.A.M., Chahoud I., Paumgartten F.J.R.

Year:

1995

Bibliographic source:

Toxicology 96, 105-113

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

(Exposure period gestation day 6 to 15; data on maternal observations restricted to body weight gain; soft tissue examinations on one-third of fetuses per litter, skeletal examinations on two-third per litter)

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Citral

EC Number:

226-394-6

EC Name:

Citral

Cas Number:

5392-40-5

IUPAC Name:

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

Cas Number:

106-26-3

Molecular formula:

C10H16O

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): citral
- Purity: 95 %

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Oswaldo Cruz Foundation Animal House breeding stock
- Housing: plastic cages
- Diet: Nuvital ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 1
- Humidity (%): 70%
- Photoperiod (hrs dark / hrs light): 12/12 hrs

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- in corn oil (Mazola)

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

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: 2 h

- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0 ] of pregnancy
Pregnancy confirmed by implantation sites shown by Salewski’s method

Duration of treatment / exposure:

gestation day 6 to 15

Frequency of treatment:

daily

Duration of test:

up to gestation day 21

Doses / concentrations

Remarks:

Doses / Concentrations:
60, 125, 250, 500, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

N=20 females (N=19 in 60 mg/kg-group)

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: day 0 and day 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day: 21
- Organs examined: no data

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 resorptions: Yes
- Number of live fetuses: Yes
- Fetal weight: individual and litter
- Sex ratio: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one-third per litter
- Skeletal examinations: Yes: two-third per litter
- Head examinations: Yes
- Fetal organ weights: heart, lungs, thymus, liver, spleen, kidneys

Statistics:

Data were analysed by one-way analysis of variance or, alternatively, by the Kruskal-Wallis test whenever the data did not fit a normal distribution. Differences between groups were tested by using two-sided either Student t-test or Mann-Whitney U-test. Proportions were analysed by the Chi-square test. In all instances statistical evaluation was performed using a MINITAB program (MTB, University of Pennsylvania, 1984), and a difference was considered statistically significant at P< 0.05.

Historical control data:

No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
Body weight/ body weight gain / body weight gain - uterus weight (for details see Table 1):
60 mg/kg bw/d:
- significant reduction of bw gain for day 6-11 (61% vs controls) and day 6-15 (73% vs controls);
- non significant reduction of bw gain for day 0-21.

125 mg/kg bw/d:
- non significant reduction in body weights (day 21);
- significant reduction of bw gain for day 6-11 (30% of control) and day 0-21(79% vs controls);

250 mg/kg bw/d:
- non significant reduction in body weights (day 21);
- significant reduction of bw gain for day 6-15 (56% vs controls) and day 0-21 (82% vs control); non significant reduction in bw gain for day 6-11
- non significant reduction in bw gain (-uterus weight);

500 mg/kg bw/d:
- non significant reduction in body weights (day 21);
- non significant reduction of bw gain for day 6-11, day 6-15 and day 0-21;
- significant reduction in bw gain (-uterus weight), (70% vs controls);

1000 mg/kg bw/d:
- significant reduction in body weights (day 21), (84% vs controls);
- significant reduction of bw gain for day 6-11 (17% vs controls), day 6-15 (47% vs controls) and day 0-21 (56% vs controls);
- significant reduction in bw gain (-uterus weight), (24% vs controls);

Gravid uterine weight (all doses refer to mg/kg bw/d; for details see Table 1):
125, 250, 1000 mg/kg: significant reduction; non-significant reduction at 500 mg/kg

Evaluation: At 125 and 250 mg/kg reduction of overall pregnancy weight gain seemed to have resulted from a decrease in gravid uterine weight. At 500 and 1000 mg/kg, the deficit in overall weight gain points to a maternal toxic effect since the difference is even more evident when gravid uterus weight is deducted from overall weight gain. The reduction in pregnancy weight gain at 60 and 125 mg/kg noted during day 6-11, must also be considered as sign of maternal toxicity, since the weight of the embryos is insignificant at this stage of gestation.

Mortality:
There is no clear relationship between treatment and a single death in the 250 mg/kg-group, 5 days after termination of treatment. Autopsy revealed no salient finding.

Effect levels (maternal animals)

1

Dose descriptor:

LOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

LOAEL

Effect level:

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

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
All doses refer to mg/kg bw/d

Implantation:
from 125 mg/kg onward: dose-dependent decrease of ratio of pregnant rats per sperm-positive rats (significant from 250 mg/kg onward);
corpora lutea graviditatis found in most of sperm-positive treated rats without implantation sites
At 125 and 1000 mg/kg: significant decrease of implantation sites per dam detected at caesarean section (for details see Table 2)
At 125 and 250 mg/kg: significant reduction of mean number of corpora lutea, no significant reduction at higher doses.

Evaluation: These findings suggest that citral at the very beginning of the treatment period impaired implantation and/or induced very early post-implantation losses which were not detected at caesarean section. Consequently, citral induced a reduction of sperm positive rats with implantation sites and the mean number of implantations per mother. However, this effect is not attributable to a decrease in the mean number of corpora lutea, because most of citral-treated females which had no implantation sites presented corpora lutea graviditatis. Citral seemed to have induced whole-litter rather than intra-litter individual losses. The overlapping with overt maternal toxicity suggested that substance-induced prenatal losses was a maternally-mediated effect.

Post-implantation losses:
60 and 125 mg/kg: significant increase of ratio of resorptions per implantations (for details see Table 2); no significant effect at higher doses.
Evaluation: Since at 250 mg/kg and higher doses there was an increase in pre- or peri-implantation losses (decrease in the proportion of sperm-positive females with implantation sites), it seems that citral-induced gestational losses occurred earlier as the dose increased.

Mean number of live fetuses (see Table 2):
from 125 mg/kg onward: significant reduction (non-significant reduction in the 500 mg/kg dose group).
Evaluation: Effect is caused by increased proportion of resorptions as well as reduced number of implantations.

Fetal body weight:
from 125 mg/kg onward: significant decrease of individual fetal body weights (significant decrease of mean litter fetal body weights from 250 mg/kg onward; See table 2)

Frequency of fetuses showing signs of delayed ossification:
from 125 mg/kg onward: significant increase (non-significant increase in the 1000 mg/kg dose group; see table 3).

Evaluation: Embryofetal development was retarded at 125 mg/kg and higher doses. This can be attributed to the citral-induced maternal toxicity.

Externally visible gross-structural abnormalities or visceral malformations (see Table 4):
from 250 mg/kg onward: significant increase of incidence of hematoma

Fetal organ weights (see Table 4):
from 125 mg/kg onward: significant increase of relative spleen weight
from 250 mg/kg onward: significant increase of absolute spleen weight


Skeletal gross-structural abnormalities (see Table 5):
125, 250 and 1000 mg/kg: significant increase of incidence of total fetuses with minor skeletal abnomalies located mainly in the skull, sternum, vertebral column and ribs
Evaluation: This effect occurring together with fetal growth retardation can be attributed to the citral-induced maternal toxicity.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Pregnancy parameters and maternal weight gain of rats treated orally with citral on day 6-15 of pregnancy

Treatment	Citral (mg/kg bw/d)
	0	60	125	250	500	1000
Treated females	20	19	20	20	20	20
Pregnant females	17	16^a	13	12^b	12	6
Pregnant/treated females (%)	85	84	65	60*	60*	30*
Non-pregnant females with CL	1	3	7	6	8	13
without CL	2	-	-	2	-	1
Maternal weight (g)
Day 0	213 ±14	221 ± 27	215 ± 33	207 ± 39	213 ± 52	210 ± 65
Day 21	326 ± 34	324 ±24	305 ± 27	299 ± 26	305 ± 36	274 ± 32*
Pregnant uterus weight (g)	74.9 ± 15.8	66.2 ±15.9	49.7 ± 22.9*	58.9 ± 12*	64.9 ± 25.3	54.1 ± 28.5*
Maternal weight gain (g)
Day 6-11	16.3 ± 7.2	10.0 ± 7.3*	10.1 ± 5.9*	12.2 ± 5.3	11.2 ± 8.7	2.7 ± 9.9*
Day 6-15	33.2 ± 9.2	24.2 ± 6.7*	28.2 ± 6.7	18.7 ± 13.3*	27.0 ± 8.7	15.7 ± 8.5*
Day 0-21	113.2 ± 27.8	102.5 ± 23.0	89.8 ± 31.9*	92.3 ± 18.2*	91.7 ± 30.2	63.0 ± 36.1*
Day 0-21 without uterus weight	38.3 ± 18.3	36.3 ± 16.4	40.0 ± 16.6	33.4 ± 11.5	26.9 ± 19.0^*	9.2 ± 27.4*

CL: corpora lutea graviditatis

^a: one pregnant female delivered on day 20

^b: one pregnant female died on day 20

Percentage of pregnant females was analysed by chi-square test and maternal weight gain was analysed by the Krusal-Wallis test followed the Mann-Whitney U-test. All other parameters were analysed by one-way analysis of variance and Student's 1-test. Values are mean ± S .D .

Asterisks indicate values significantly different from controls: * P < 0.05

Table 2: Effects of citral on reproductive parameters assessed on day 21 of gestation

Treatment	Citral (mg/kg bw)
	0	60	125	250	500	1000
Corpora lutea	13.0 ± 2.2	12.0 ± 2.2	10.3 ± 2.8*	10.9 ± 2.1*	11.5 ± 2.9	11.8 ± 2.9
Implantation sites	13.2 ± 1.8	12.4 ± 2.0	10.2 ± 4.6*	11.2 ± 2.6	11.7 ± 3.6	9.2 ± 4.7*
Resorptions/implantations (%)	7.2	12.9*	15.8*	8.9	5.1	7.3
Live fetuses	12.2 ± 2.6	10.8 ± 2.9	8.6 ± 4.3*	10.2 ± 2.2*	11.1 ± 4.3	8.5 ± 4.4*
Fetal weight (g)
(individual)	4.75 ± 0.46	4.68 ± 0.40	4.24 ± 0.51*	4.25 ± 0.43*	4.53 ± 0.54*	4.29 ± 1.0*
(litter)	4.73 ± 0.32	4.69 ± 0.26	4.45 ± 0.74	4.27 ± 0.38*	4.52 ± 0.39	4.38 ± 0.95
Sex ratio (m/f)	102/106	83/80	60/52	42/60	50/61	32/19*

Fetal body weight was analyzed by taking either the individual fetus weight or litter mean fetal weight as a statistical unit.

Number of corpora lutea and implantation sites were analyzed by the Kruskal-Wallis test followed by the Mann-Whitney U-test; number of live fetuses and fetal body weight were analyzed by one-way analysis of variance followed by Student's t-test. Sex ratio and percentage of respirations were analyzed by Chi-square test. Values are mean ± S .D .

Asterisks indicate values significantly different from controls: * P < 0 .05).

Table 3: Occurrence of delayed ossification in rat fetuses treated orally with citral on days 6-15 of pregnancy

Treatment	Citral (mg/kg bw)
	0	60	125	250	500	1000
Fetuses examined	133	110	78	71	76	29
Fetuses with signs of delayed ossification (%)	18.0	13.6	73.1*	87.3*	46.0*	24.1
% of fetuses with signs of delayed ossification in:
Skull bones	3.7	2.7	24.3*	21.1*	7.9	10.3
Caudal vertebrae	12.0	10.9	24.3*	28.2*	9.2	6.9
Ribs	0	0	1.3	2.8	1.3	3.4
Forelimbs	6.0	2.7	59.0*	85.9*	36.8*	13.8
Hindlimbs	12.8	10.0	46.5*	50.7*	21.0	13.8

Signs of delayed ossification : not ossified (whole bone not stained), poorly ossified (whole bone is poorly stained), irregular spongy bones.

Chi-square test: values significantly different from controls are indicated by an asterisk: * P < 0.05

Table 4: Occurrence of externally visible anomalies and spleen weight changes in fetuses of rats treated orally with citral on days 6-15 of pregnancy

Treatment	Citral (mg/kg bw)
	0	60	125	250	500	1000
External examination (no of fetuses)	208	163	112	102	111	51
Hematoma (%)	3 (1.4)	1 (0.6)	4 (3.6)	7 (6.9)*	7 (6.3)*	7 (13.7)*
Tongue protusion	0	0	1	0	0	0
Visceral examination (no of fetuses)	75	53	34	31	35	15
Fetal weight (g)	5.08 ± 0.51	5.07 ± 0.53	4.58 ± 0.54*	4.80 ± 0.46	4.75 ± 0.74	4.75 ± 1.02
Spleen weight (mg)	5.2 ± 1.2	5.4 ± 1.5	5.4 ± 1.0	6.1 ± 1.1*	7.1 ± 2.3*	6.2 ± 1.7*
Spleen weight (mg)/ Fetal weight (g)	1.1 ± 0.26	1.16 ± 0.21	1.28 ± 0.22*	1.41 ± 0.22*	1.53 ± 0.35*	1.45 ± 0.31*

Fetuses fixed in 5% formalin solution; values are mean ± S .D.

Fetal body weight and spleen weight were analysed by one-way analysis of variance followed by Student's t-test ; spleen weight per fetal weight ratio was analysed by the Kruskal-Wallis test followed by the Mann-Whitney U-test; and percentage of fetuses with hematoma was analysed by the Chi-square test.

Asterisks indicate values significantly different from controls: * P < 0 .05)

Table 5: Occurrence of skeletal anomalies in rat fetuses treated orally with citral on days 6-15 of pregnancy

Treatment	Citral (mg/kg bw)
	0	60	125	250	500	1000
Fetuses examined	133	110	78	71	76	29
Total fetuses with skeletal abnormalies (%)	4.5	5.4	11.5*	15.5*	5.3	13.8*
Percentage of fetuses showing abnormalies in:
Skull	1.5	2.7	5.1	1.4	1.3	5.4
Os interparietale (bent)	0	0	0	1.4	0	0
Os basisphenoid (irregular shape)	1.5	2.7	3.8	0	1.3	5.4
Os hamulus (smaller)	0	0	1.3	0	0	0
Sternum	0.7	0	5.1	4.2	1.3	8.1
two ossification centra	0	0	0	0	0	2.7
dislocated	0.7	0	5.1	4.2	1.3	5.4
Vertebral column	2.8	0	0	5.6	1.3	0
Atlas longer	0.7	0	0	1.4	1.3	0
Lumbar vertebrae
Additional	0.7	0	0	0	0	0
Dumb bell	0.7	0	0	0	0	0
Thoratic vertebrae (dumb bell)	0.7	0	0	4.2	0	0
Ribs	2.2	2.7	1.3	4.2	1.3	0
missing	0	0	0	2.8	1.3	0
Extra
lumbar	0.7	0.9	0	0	0	0
cervical	1.5	1.8	1.3	1.4	0	0

Dumb bell: ossification centre dumb bell shaped.

Comparisons were made by the Chi-square test.

Asterisks indicate values significantly different from controls: * P < 0 .05)