Litsea cubeba, ext.

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1995 - September 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with minor restrictions.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1995 - September 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with minor restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(exposure 98 days, partly other organ weights and other clinical chemistry parameters, no urinalysis or ophthalmological examination)

GLP compliance:

yes

Remarks:

FDA GLP regulations

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services, Germantown, N.Y.
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: ca. 80 g
- Fasting period before study: no
- Housing: 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11 to 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C (calculated from 72° +- 3° F)
- Humidity (%): 50% +- 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: June 5 or 6, 1995 To: September 5 or 6, 1995

Route of administration:

oral: feed

Vehicle:

other: microcapsules loaded with citral

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 to 4 weeks
- Mixing appropriate amounts with nonirradiated NTP-2000 feed: final concentrations of 3900, 7800, 15600, or 31300 ppm citral were achieved by adding loaded and/or placebo microcapsules at a total concentration of 10% microcapsules
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: microcapsules prepared from food-grade sugar and starch were loaded with citral to prevent loss of test substance
- Concentration in vehicle: 31.3 % citral (analytical method GC)
- Lot/batch no.: 20295

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

periodic verification by GC

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuously

Remarks:

Doses / Concentrations:
3900, 7800, 15600, 31300 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
ca. 345, 820, 1785, and 1586 mg/kg bw
Basis:
other: actual ingested by male rats

Remarks:

Doses / Concentrations:
ca. 335, 675, 1330, and 1215 mg/kg bw
Basis:
other: actual ingested by female rats

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:based on pre-study of Dieter et al., 1993

Positive control:

not necessary

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly, and at termination of study

FOOD CONSUMPTION AND COMPOUND INTAKE : weekly
- 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: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 4, 22 and after 14 weeks of treatment
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No data
- How many animals: 10 per dose
- Parameters: erythrocyte, reticulocyte, platelet counts, hematocrit, hemoglobin concentration, erythrocyte morphology, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, leukocyte counts and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 4, 22 and after 14 weeks of treatment
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No data
- How many animals: 10 per dose
- Parameters: urea nitrogen, creatinine, total protein, albgumin, alanine aminotransferase, alkaline phosphatase, creatinine kinase, sorbitol dehydrogenase, bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
heart, right kidney, liver, lung, right testis, thymus

HISTOPATHOLOGY: Yes
Complete histopathology performed on untreated controls, vehicle controls, dose groups with food concentrations of 15,600, or 31,300 ppm;
examined tissues: gross lesions and tissue masses, adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart and aorta, large intestine, small intestine, kidney, liver, lung and mainstem bronchi, lymph nodes , mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach, testis with epididymis and seminal vescicles, thymus, thyroid gland, trachea, urinary bladder, uterus
In addition, examination of bone marrow and forestomach (both sexes) and of kidney (male rats) of lower dose groups to find a NOEL.
Specific staining of hyaline droplets in renal tubules with H&E and Mallory Heidenhain stains

Statistics:

Body weights: Williams' or Dunnett's test
Hematology or clinical chemistry data: Dunn's or Shirley's test

Details on results:

CLINICAL SIGNS AND MORTALITY
31300 ppm: all rats killed moribund in second week of treatment; clinical signs were listlessness, hunched posture, absent or slow paw reflex, dull eyes
other dose groups: all animals survived to the end of the study, no clinical signs reported

BODY WEIGHT AND WEIGHT GAIN (for details see Table 1)
all dose groups: significant decrease of final mean body weight; signifcant decrease of body weight gain in all dosed males
from 7800 ppm: signifcant decrease of body weight gain in females

FOOD CONSUMPTION AND COMPOUND INTAKE (for details see Table 1)
from 15600 ppm: decreased food consumption during first study week resulting in lowered average daily doses of the rats of the 31,300 ppm-group which were killed untimely

HAEMATOLOGY
Changes tended to be transient and to improve with increased duration of exposure. They were in general not observed after day 22:
day 4: from 7800 ppm significant increases in hematocrit values, hemoglobin concentrations, erythrocyte and platelet counts; significant decreases in mean cell volumes, mean cell hemoglobin values, reticulocyte and nucleated erythrocyte counts
day 22: 15600 ppm significant decreases in mean cell volumes and mean cell hemoglobin values
week 14: male rats: significant increase of mean cell volume; female rats: significant decrease of mean cell volumes, significant increase of erythrocyte counts
Evaluation - no effects: Changes in erythrocyte and platelet counts were consistent with known physiologic responses related to decreased food and water consumption, and were not considered to be a direct cause of a toxic action of citral.

CLINICAL CHEMISTRY
Generally, the changes tended to be transient and to improve with increased duration of exposure:
day 4: from 3900 ppm: both sexes: significant increase of urea nitrogen; male rats: significant increase of albumin
from 15600 ppm: both sexes: significant decrease of alkaline phosphatase; male rats: significant increase of total protein;
female rats: significant increase of bile acids
day 22: from 3900 ppm: male rats: significant increase of urea nitrogen
from 7800 ppm: female rats: significant increase of alkaline phosphatase
15,600 ppm: both sexes: significant increase of albumin; female rats: significant increase of urea nitrogen and bile acids
week 14: 7800 ppm: male rats: significant increase of albumin
from 7800 ppm: female rats: significant increase of alkaline phosphatase
15600 ppm: male rats: significant increase of urea nitrogen
Evaluation - no effects: Decreased food consumption (see Table 1) and possibly water consumption (no data available) were discussed as causes of physiological responses leading to the changes of serum biochemical parameters. Alterations in albumin, total protein, and urea nitrogen concentrations may be related to possible dehydration or to decreased glomerular filtration rates due to renal damage. The decreases in alkaline phosphatase activity may reflect a loss of circulating intestinal isoenzyme fraction related to decreased feed consumption. In females, bile acid concentration and alkaline phosphatase were increased at 15600 ppm and 31300 ppm. In general, these parameters are considered indicators of bile stasis and would suggest that a cholestatic event may have occurred. However, alterations were of minimal severity and transient, and there was no histopathologic evidence of cholestasis, suggesting that these changes were not biologically significant.

ORGAN WEIGHTS (for details see Table 1)
Evaluation - no effects: Changes were minor with significant decreases of absolute organ weights and significant increases of relative organ weights. These were considered to be related to the significant decreases of final body weight and to be of no biological relevance or toxicologically significance.

GROSS PATHOLOGY
No exposure-related changes

HISTOPATHOLOGY: NON-NEOPLASTIC
Forestomach:
at 31300 ppm after premature sacrifice in the second week epithelial hyperplasia (2/10 m, 4/10 f) and hyperkeratosis (2/10 m, 4/10 f) , with thickening of stratified squamous epithelium and of the cornified superficial layer of the mucosa were observed, no signs of inflammation; significant increase in f, p<=0.01
Bone marrow:
at 15600 ppm increased incidence of minimal grade of atrophy in 7/10 m and 8/10 f, p<=0.01;
31,300 ppm: all males showed atrophy and hemorrhage; incidences in f: atrophy 4/10, p<=0.05, hemorrhage 9/10, p<=0.01
Thymus:
at 31300 ppm atrophy in 5/10 m and 4/10 f, p<=0.05
Kidney:
minimal to mild nephropathy present in males of all dose groups with incidences of 3/10, 10/10, 8/10, 0/10 (premature termination of high dose group), vehicle control 0/10, with significant increases at 7800 and 15600 ppm, p<=0.01; changes characterized by foci of regenerative epithelium, occasional eosinophilic casts, peritubular mononuclear inflammation, and dilated tubules;
Granular casts in renal tubules in males of all dose groups with incidences of 3/10, 10/10, 10/10, 0/10 (premature termination of high dose group), vehicle control 0/10, with significant increases at 7800 and 15600 ppm, p<=0.01; changes characterized by few and scattered granular casts within the outer strip of the medulla, dilated tubules filled with granular eosinophilic material presumed to be proteinacious material and cellular debris; no apparent increase in the amount of hyaline droplets.
Testes:
aspermia in all males at 31000 ppm
Evaluation: Findings in the forestomach, thymic atrophy, and aspermia in the testes were limited to the high dose group that had to be sacrificed in the second study week due to moribundity. It was not clear if the bone marrow lesions in the same dose group were a direct effect of citral toxicity or due to inanition. At the lower dose of 15600 ppm minimal atrophy of bone marrow without accompanying hemorrhage was considered a borderline lesion. Concerning the renal changes in male rats of all dose groups, the presence of granular casts and exacerbation of spontaneous nephropathy would be suggestive of an alpha2µ globulin nephropathy. However, it was considered that renal lesions were not mediated by alpha2µ globulin as there was no increased incidence of hyaline droplets.

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: decrease of final body weight and body weight change / dose corresponds to 3900 ppm in diet

Dose descriptor:

LOAEL

Effect level:

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

Sex:

female

Basis for effect level:

other: decrease of final body weight / dose corresponds to 3900 ppm in diet

Critical effects observed:

not specified

Table 1: Survival, body and organ weight data, and feed consumption in the 14-day feeding study with citral

Concentration	Survival	Final body weight (g)	Weight change (g)	Feed consumption (g/animal/d)		Organ weight changes ^e
				Week 1	Week 14	absolute	relative
Males
Vehicle control	10/10	336 +-6	255 +-6	15.4	18.7	-	-
3900 ppm	10/10	318 +-6^a	238 +-5^b	15.9	19.9	-	kidney^ctestis^c
7800 ppm	10/10	292 +-4^b	208 +-3^b	15.1	20.1	heart^blung^athymus^b	kidney^cliver^ctestis^c
15600 ppm	10/10	247 +-4^b	163 +-4^b	8.4	15.6	heart^bliver^blung^bthymus^b	kidney^cliver^ctestis^c
31300 ppm	0/10	-	-	4.0	-	-	-
Females
Vehicle control	10/10	190 +-4	108 +-4	12.8	10.7	-	-
3900 ppm	10/10	180 +-4^a	101 +-4	11.6	9.6	-	kidney^c
7800 ppm	10/10	181 +-2^a	97 +-2^a	11.8	10.8	-	kidney^c
15600 ppm	10/10	166 +-2^b	82 +-2^b	6.5	10.2	heart^c	kidney^cliver^d
31300 ppm	0/10	-	-	4.7	-	-	-

Significant changes in comparison to vehicle control:

^a significant decrease, p<=0.05; ^b significant decrease, p<=0.01; ^c significant increase, p<=0.01; ^d significant increase, p<=0.05

^e The relevance of observed organ weight changes is discussed in "Details on results"

Conclusions:

Based on the results of the study, the LOAELs of Citral were set at 345 and 335 mg/kg bw (3900ppm) for male and female rats based on decreased body weight.

Executive summary:

The substance Citral (Reaction mass of (E)-3,7-dimethylocta-2,6-dienal and (Z)-3,7-dimethylocta-2,6-dienal) has been tested in a study similar to OECD TG 408. Groups of male and female F344/N rats were exposed to microencapsulated citral (greater than 96% pure) in feed for 14 weeks. Dose used in rats were equivalent to 345-1,585 mg citral/kg body weight for males and 335- 2,125 mg/kg for females.

Effects observed: Lower mean body weights, and feed consumption. Males and females in the high dose group exhibited listlessness, hunched posture, absent or slow paw reflex, and dull eyes. Some evidence of forestomach epithelial hyperplasia and hyperkeratosis, bone marrow atrophy and hemorrhage, and nephrotoxicity was observed.

Based on these results the LOAEL of rats at 14 weeks was set at 345 and 335 mg/kg bw (3900ppm) based on decreased body weight.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1995 - September 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

(complete histopathology for all dose groups; no parameters of hematology, clinical chemistry or urinalyses measured)

GLP compliance:

yes

Remarks:

FDA GLP regulations

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services, Germantown, N.Y.
- Age at study initiation: 6 weeks
- Weight at study initiation: males ca. 20 g, females ca. 16.5 g
- Fasting period before study: no
- Housing: males inidividually, females 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 to 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C (calculated from 72° +- 3° F)
- Humidity (%): 50% +- 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: June 19 or 20, 1996 To: June 15-19 (males) or June 22-24 (females), 1998

Route of administration:

oral: feed

Vehicle:

other: microcapsules loaded with citral

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 weeks
- Mixing appropriate amounts with nonirradiated NTP-2000 feed: final concentrations of 0, 500, 1000, or 2000 ppm citral were achieved by adding loaded and/or placebo microcapsules at a total concentration of 1.25% microcapsules
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: microcapsules prepared from food-grade sugar and starch were loaded with citral to prevent loss of test substance
- Concentration in vehicle: 31.9 % citral (analytical method GC)
- Lot/batch no.: MRI 020196MC

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

periodic verification by GC

Duration of treatment / exposure:

104-105 weeks

Frequency of treatment:

continuously

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
ca. 0, 60, 120, 260 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on 14-week pre-study described in same study report

Positive control:

Not necessary

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: day 8, 36, and every 4 weeks thereafter, at end of study

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 8, 36, and every 4 weeks thereafter, at end of study

FOOD CONSUMPTION AND COMPOUND INTAKE : weekly
- Food consumption for each cage determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, approximately every 4 weeks for a 1-week period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Complete histopathology performed on all mice:
examined tissues: gross lesions and tissue masses, adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder, heart and aorta, large intestine, small intestine, kidney, liver, lung and mainstem bronchi, lymph nodes , mammary gland (females only), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach, testis with epididymis and seminal vescicles, thymus, thyroid gland, trachea, urinary bladder, uterus

Statistics:

Probability of survival: product-limit procedure of Kaplan and Meier
dose-related effects on survival: Cox's method, Tarone's life table test
Body weight data: parametric multiple comparison procedures of Dunnett and Williams
Incidences of non-neoplastic lesions: Poly-3 test (Bailer and Portier, 1988)

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival of all dosed groups similar to vehicle control groups; no clinical findings attributable to citral exposure

BODY WEIGHT AND WEIGHT GAIN (for details see Table 1)
Mean body weights were generally less compared to those of the vehicle controls in the following dose groups and study periods:
females: at 500 ppm from week 30-102, at 1000 ppm from week 14-102, at 2000 ppm during whole study period;
males: at 1000 ppm from year 2, at 2000 ppm during whole study period.
Final body weights (101 w): females: 91%, 88% and 78% of controls; males: 93%, 91% and 84% of controls for 500, 1000, 2000 ppm respectively.

FOOD CONSUMPTION AND COMPOUND INTAKE (for details see Table 1)
Comparable to vehicle control

PATHOLOGY: NON-NEOPLASTIC
- Oral mucosa: Inflammation and ulceration
Incidences: present in all groups including vehicle controls with significantly increased incidences in all exposed female mice and 2000 ppm male mice (for incidences see Table 2)
Location: areas of inflammation and ulceration directly medial to the molar teeth in most cases; ulceration almost always located at the points were hair shafts penetrated the oral mucosa
Characterization of inflammation: minimal to mild severity (see Table 2), accumulation of mixed inflammatory cells within and just beneath the oral mucosa adjacent to the medial aspect of the teeth; in a majority of cases hair shafts present in the inflamed areas and appeared to have penetrated the tooth socket
Characterization of ulceration: minimal to mild severity (see Table 2), focal areas with loss of mucosa
Evaluation: inflammation and ulceration were considered to be secondary to embedded hair shafts. The same lesions, with similar severity, were present in vehicle controls. Thus, lesions in the oral mucosa were considered probably not a direct toxic effect of citral, but citral may have exacerbated the secondary inflammatory response in females. The significance of this effect is unknown.

- Adrenal cortex: focal hyperplasia, significant increase in 2000 ppm male mice (for incidences see Table 2)
Evaluation: Incidences were very low for this common background lesion and are therefore not considered to reflect a toxic response to citral exposure.

- Bone: fibrosis, significant increase in 500 and 1000 ppm female mice (see Table 2)
Evaluation: The significance of this effect without dose-relationship is unknown.

- Kidney: minimal nephropathy with significant increase in 2000 ppm females and minimal renal tubule mineralization with significant increase in 500 and 1000 ppm females (for incidences see Table 2)
Evaluation: The toxicological significance of increased incidences of these minimal changes also present in vehicle controls is unclear.

PATHOLOGY: NEOPLASTIC
Equivocal evidence of treatment-related induction of malignant lymphoma in female mice: Findings are presented in Section 7.7 in detail.

Dose descriptor:

LOAEL

Effect level:

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

Sex:

female

Basis for effect level:

other: reduction of body weights / dose corresponding to 500 ppm in diet

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: reduction of body weights/ dose corresponding to 1000 ppm in diet

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: dose corresponding to 500 ppm in diet

Critical effects observed:

not specified

Table 1: Body weight data for mice in the 2-year feed study of citral

Sex	Timepoint	Vehicle control	500 ppm		1000 ppm		2000 ppm
		Av wt (g)	Av wt (g)	Rel wt (%)^a	Av wt (g)	Rel wt (%)^a	Av wt (g)	Rel wt (%)^a
m	Mean w 1-13	25.3	25.2	100	24.7	98	24.2	96
	Mean w 14-52	40.0	39.2	98	38.2	96	36.7	92
	Mean w 53-101	46.6	44.9	96	43.5	93	40.5	87
	Final w 102	45.9	42.8	93	41.6	91	38.6	84
f	Mean w 1-13	21.1	20.9	99	21.0	100	20.5	97
	Mean w 14-52	35.3	33.1	94	32.7	93	30.7	87
	Mean w 53-101	44.3	41.1	93	39.6	89	36.3	82
	Final w 101	47.1	42.9	91	41.4	88	36.6	78

^a weight as % of control group’s weight

Table 2: Incidences of selected non-neoplastic lesions of the oral mucosa in mice in the 2 -year feed study of citral

Type of lesion	Vehicle control		500 ppm		1000 ppm		2000 ppm
	Incidence	Severity grade^c	Incidence	Severity grade^c	Incidence	Severity grade^c	Incidence	Severity grade^c
Males
Oral mucosa, inflammation	12/50	1.8	16/50	1.9	21/50	1.9	21/50^a	1.5
Oral mucosa, ulcer	9/50	1.8	8/50	1.6	12/50	1.4	10/50	1.6
Adrenal cortex, focal hyperplasia	0/50		3/50		2/50		5/50^d
Females
Oral mucosa, inflammation	14/49	1.4	32/50^b	1.9	35/50^b	1.8	32/50^b	1.5
Oral mucosa, ulcer	6/49	1.2	15/50^b	1.9	22/50^b	1.6	15/50^a	1.5
Bone, fibrosis	11/49		22/50^d		21/50^d		18/50
Kidney, nephropathy	9/49	1.0	16/50	1.0	15/50	1.2	17/50^a	1.0
Kidney, renal tubule mineralization	4/50	1.0	14/50^a	1.0	18/50^b	1.0	6/50	1.2

Significant changes in comparison to vehicle control: ^a significant increase, p<=0.05; ^b significant increase, p<=0.01; ^d significant increase, p not specified; relevance of findings is discussed in "Details on results"

^c Grading of severity: 1=minimal, 2=mild

Conclusions:

Based on the results of the study, the LOAELs of Citral were set at 120 and 60 mg/kg bw/day (1000 and 500 ppm) for male and female rats based on decreased body weight.

Executive summary:

The substance Citral (Reaction mass of (E)-3,7-dimethylocta-2,6-dienal and (Z)-3,7-dimethylocta-2,6-dienal) has been tested in a study similar to OECD TG 453. Groups of male and female B6C3 mice were exposed to microencapsulated citral (greater than 96% pure) in feed for 2 years. Dose used in the 2 year study were 60 - 260 mg/kg to males and females.

Effects observed: Lower mean body weights in the treated group. The incidences of malignant lymphoma occurred with a positive trend in female mice, and the incidence in 2,000 ppm females was significantly greater than that in the vehicle control group. Tissues most commonly affected by malignant lymphoma were the spleen, mesenteric lymph node, thymus, and, to a lesser extent, the ovary.

Based on these results the LOAELs were determined to be 120 and 60 mg/kg bw/day (1000 and 500 ppm) for males and females respectively, based on reduced body weights.

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1995 - September 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

(complete histopathology of all dose groups; no parameters of hematology, clinical chemistry or urinalyses measured)

GLP compliance:

yes

Remarks:

FDA GLP regulations

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services, Germantown, N.Y.
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: males ca. 120 g, females ca. 100 g
- Fasting period before study: no
- Housing: 2 or 3 males, or 5 females per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 to 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C (calculated from 72° +- 3° F)
- Humidity (%): 50% +- 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: June 6 or 7, 1996 To: June 1-5 (males) or June 8-10 (females), 1998

Route of administration:

oral: feed

Vehicle:

other: microcapsules loaded with citral

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 weeks
- Mixing appropriate amounts with nonirradiated NTP-2000 feed: final concentrations of 0, 1000, 2000 or 4000 ppm citral were achieved by adding loaded and/or placebo microcapsules at a total concentration of 1.25% microcapsules
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: microcapsules prepared from food-grade sugar and starch were loaded with citral to prevent loss of test substance
- Concentration in vehicle: 31.9 % citral (analytical method GC)
- Lot/batch no.: MRI 020196MC

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

periodic verification by GC

Duration of treatment / exposure:

104-105 weeks

Frequency of treatment:

continuously

Dose / conc.:

0 ppm

Remarks:

0 mg/kg bw/day

Dose / conc.:

1 000 ppm

Remarks:

50 mg/kg bw/day (nominal)

Dose / conc.:

2 000 ppm

Remarks:

100 mg/kg bw/day (nominal)

Dose / conc.:

4 000 ppm

Remarks:

210 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on 14-week pre-study described in same study report

Positive control:

not necessary

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: day 8, 33, and every 4 weeks thereafter, at end of study

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 8, 33, and every 4 weeks thereafter, at end of study

FOOD CONSUMPTION AND COMPOUND INTAKE : weekly
- Food consumption for each cage determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, approximately every 4 weeks for a 1-week period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: No

HISTOPATHOLOGY: Yes
Complete histopathology performed on untreated controls, vehicle controls, and all dosed animals:
examined tissues: gross lesions and tissue masses, adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart and aorta, large intestine, small intestine, kidney, liver, lung and mainstem bronchi, lymph nodes, mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach, testis with epididymis and seminal vescicles, thymus, thyroid gland, trachea, urinary bladder, uterus

Statistics:

Probability of survival: product-limit procedure of Kaplan and Meier
dose-related effects on survival: Cox's method, Tarone's life table test
Body weight data: parametric multiple comparison procedures of Dunnett and Williams
Incidences of non-neoplastic lesions: Poly-3 test (Bailer and Portier, 1988)

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: decrease of mean body weight; dose corresponds to 4000 ppm in diet

Key result

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: dose corresponds to 2000 ppm in diet

Critical effects observed:

not specified

Table 1: Body weight data for rats in the 2-year feed study of citral

Sex	Timepoint	Vehicle control	1000 ppm		2000 ppm		4000 ppm
		Av wt (g)	Av wt (g)	Rel wt (%)	Av wt (g)	Rel wt (%)	Av wt (g)	Rel wt (%)
m	Mean w 1-13	249	248	100	244	98	241	97
	Mean w 14-52	440	436	99	429	98	418	95
	Mean w 53-101	483	478	99	473	98	453	94
	Final w 101	479	470	98	461	96	440	92
f	Mean w 1-13	157	155	99	155	99	151	96
	Mean w 14-52	232	227	98	227	98	216	93
	Mean w 53-101	311	301	97	299	96	270	87
	Final w 101	335	326	97	323	96	298	89

Av wt: average weight

Rel wt: relative weight as % of the control group's weight

Table 2: Incidences of non-neoplastic lesions in the 2 -year feeding study with citral

	Vehicle control	1000 ppm	2000 ppm	4000 ppm
Type of lesion	Incidence	Incidence	Incidence	Incidence
Males
Kidney, renal tubule mineralization	42/50	45/50	48/50	50/50
Females
Adrenal cortex, angiectasis	1/50	1/50	3/50	10/50*

Significant changes in comparison to vehicle control:

^* significant increase, level of significance not specified

The evaluation of observed effects is discussed in "Details on results"

Conclusions:

Based on the results of the study, the LOAEL and NOAEL were set at 210 mg/kg bw (4000 ppm) and 100 mg/kg bw (1000ppm) for males and females respectively, based on decreased body weight.

Executive summary:

The substance Citral (Reaction mass of (E)-3,7-dimethylocta-2,6-dienal and (Z)-3,7-dimethylocta-2,6-dienal) has been tested in a study similar to OECD TG 453. Groups of male and female F344/N rats were exposed to microencapsulated citral (greater than 96% pure) in feed for 2 years. Doses used in rats in the 2 year study were 50 - 210 mg/kg to males and females.

Effects observed: Higher survival of all exposed groups of males compared to controls. Lower mean body weights in exposed groups. No neoplasms or non-neoplastic lesions were attributed to exposure to citral.

Based on these results the LOAEL and NOAEL were set at 210 mg/kg bw (4000 ppm) and 100 mg/kg bw (1000ppm) respectively for males and females, based on decreased body weights.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2003

Reference 2

Reference Type:

publication

Title:

Toxicology and Carcinogenesis Studies of Microencapsulated Citral in Rats and Mice

Author:

Ress NB, Hailey JR, Maronpot RR, Bucher JR, Travlos GS, Haseman JK, Orzech DP, Johnson JD, Hejtmancik MR

Year:

2003

Bibliographic source:

Toxicol. Sci., 71, 198-206

Reference 3

Reference Type:

other: abstract

Title:

TR-505 Toxicology and Carcinogenesis Studies of Citral (Microencapsulated) (CAS No. 5392-40-5) in F344/N Rats and B6C3F1 Mice (Feed Studies) - Draft Abstract

Author:

National Toxicology Program NTP

Year:

2003

Bibliographic source:

http://ntp-server.niehs .nih .gov/htdocs/LT-studies/tr505 .html

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

(partly other organ weights and clinical chemistry parameters, no urinalysis or ophthalmological examination)

GLP compliance:

yes

Remarks:

FDA GLP regulations

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

Cas Number:

106-26-3

Molecular formula:

C10H16O

IUPAC Name:

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

Details on test material:

- Name of test material (as cited in study report): Citral
- Physical state: colourless liquid
- Analytical purity: ca. 97.6%
- Impurities (identity and concentrations): seven substances corresponding to 2.4 % in total
- Isomers composition: ratio geranial:neral = 2:1
- Lot/batch No.: 06930PG
- Expiration date of the lot/batch: no data
- Stability under test conditions: no degradation of the neat chemical after storage for 2 weeks at temperatures up to 60°C when protected from light; no loss of citral from the microcapsules during 14 weeks

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Laboratory Animals and Services, Germantown, N.Y.
- Age at study initiation: 6 weeks
- Weight at study initiation: males ca. 20 g, females ca. 16.5 g
- Fasting period before study: no
- Housing: males inidividually, females 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 to 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C (calculated from 72° +- 3° F)
- Humidity (%): 50% +- 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12


IN-LIFE DATES: From: June 7 or 8, 1995 To: September 7 or 8, 1995

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: microcapsules loaded with citral

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 to 4 weeks
- Mixing appropriate amounts with nonirradiated NTP-2000 feed: final concentrations of 3,900, 7,800, 15,600, or 31,300 ppm citral were achieved by adding loaded and/or placebo microcapsules at a total concentration of 10% microcapsules
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: microcapsules prepared from food-grade sugar and starch were loaded with citral to prevent loss of test substance
- Concentration in vehicle: 31.3 % citral (analytical method GC)
- Lot/batch no.: 20295

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

periodic verification by GC

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

continuously

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
3900, 7800, 15600, or 31300 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
745, 1840, 3915, and 8110 mg/kg bw/d
Basis:
other: actual ingested by male mice

Doses / concentrations 3

Remarks:

Doses / Concentrations:
790, 1820, 3870, and 7550 mg/kg bw/d
Basis:
other: actual ingested by female mice

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on pre-study of Dieter et al., 1993

Positive control:

Not necessary

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: initially, weekly, and at termination of study

FOOD CONSUMPTION AND COMPOUND INTAKE : weekly
- 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: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 4, 22 and after 14 weeks of treatment
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No data
- How many animals: 10 per dose
- Parameters: erythrocyte, reticulocyte, platelet counts, hematocrit, hemoglobin concentration, erythrocyte morphology, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, leukocyte counts and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 4, 22 and after 14 weeks of treatment
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No data
- How many animals: 10 per dose
- Parameters: urea nitrogen, creatinine, total protein, albgumin, alanine aminotransferase, alkaline phosphatase, creatinine kinase, sorbitol dehydrogenase, bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
heart, right kidney, liver, lung, right testis, thymus

HISTOPATHOLOGY: Yes
Complete histopathology performed on untreated controls, vehicle controls, dose groups with food concentrations of 15,600, or 31,300 ppm;
examined tissues: gross lesions and tissue masses, adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder, heart and aorta, large intestine, small intestine, kidney, liver, lung and mainstem bronchi, lymph nodes , mammary gland (females only), nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach, testis with epididymis and seminal vescicles, thymus, thyroid gland, trachea, urinary bladder, uterus
In addition, examination of forestomach and of ovaries of lower dose groups to find a NOEL

Statistics:

Body weights: Williams' or Dunnett's test
Hematology or clinical chemistry data: Dunn's or Shirley's test

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
31300 ppm: 4 males killed moribund in second week of treatment
clinical signs: at 15600 and 31300 ppm animals were generally thin and appeared lethargic; at 7800 ppm few males were also thin
other dose groups: all animals survived to the end of the study

BODY WEIGHT AND WEIGHT GAIN (for details see Table 1)
all dose groups: significant dose-dependent decreases of final mean body weight and of body weight gain
31300 ppm: final weight < initial weight

FOOD CONSUMPTION AND COMPOUND INTAKE (for details see Table 1)
from 7800 ppm: females showed decreased food consumption during first study week
all doses: by the end of the study, food consumption was greater than by vehicle control
Evaluation: increased food consumption may have been due to the mice scattering feed, an indication of poor palatability

HAEMATOLOGY
week 14: from 15600 ppm: significant dose-dependent decreases of leukocyte and lymphocyte counts; in male mice lymphocyte counts were already decreased at 3900 and 7800 ppm without a dose-dependency, however, some other parameters were increased at single dosages showing no biological relevance.
Evaluation: These changes together with marked suppression in mean body weights may reflect a physiological response consistent with a stress-related and/or corticosteroid-induced lymphopenia.

ORGAN WEIGHTS
Significant decreases of absolute organ weights, increases of relative organ weights (for details see Table 1)
Evaluation: differences in organ weights between exposed mice and vehicle controls reflected body weight differences and were not toxicologically significant

GROSS PATHOLOGY
No exposure-related changes

HISTOPATHOLOGY: NON-NEOPLASTIC
Forestomach:
from 15,600 ppm: wall of forestomach of many males and females (incidences not given) variably thickened (2 to 5times normal), mucosa (squamous epithelium) and submucosa were often rugose; additionally, minimal hyperkeratosis of epithelium being insignificant
Evaluation: Although thickened, all three main components (mucosa, submucosa, and muscle) appeared proportional to each other and to those of control animals. Therefore alteration considered to be the result of a contradicted stomach rather than a pathological alteration.
Ovaries:
from 15,600 ppm: increased incidences of ovarian atrophy characterized by absence of or reduction in the number of corpora lutea with no effect on primary, secondary, or antral follicles; incidences 0/10, 0/10, 7/10, 10/10, vehicle control 0/10
Evaluation: NTP Pathology Working group considered these lesions most probably to represent hypoplasia being most likely a secondary effect due to the poor condition of female mice in these dose groups.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: reduced final body weight and body weight change / dose corresponds to 3900 ppm in diet

2

Dose descriptor:

LOAEL

Effect level:

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

Sex:

female

Basis for effect level:

other: reduced final body weight and body weight change / dose corresponds to 3900 ppm in diet

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Survival, body weights, feed consumption, and organ weights of mice in the 14-week feed study of citral

Concentration	Survival	Final body weight (g)	Weight change (g)	Feed consumption (g/animal/d)		Organ weight changes ^e
				Week 1	Week 14	absolute	relative
Males
Vehicle control	10/10	33.2 +- 0.8	12.6 +- 0.7	4.4	4.5	-	-
3900 ppm	10/10	28.1 +- 0.6^b	7.9 +- 0.6^b	4.6	5.0	-	kidney^cliver^cthymus^d
7800 ppm	10/10	25.6 +- 0.6^b	5.2 +- 0.4^b	4.3	5.9	kidney^bthymus^a	heart^ckidney^cliver^clung^d
15600 ppm	10/10	21.3 +- 0.6^b	1.3 +- 0.5^b	4.0	6.2	kidney^bliver^btestis^bthymus^a	heart^ckidney^cliver^clung^cthymus^c
31300 ppm	6/10	17.1 +- 0.4^b	-2.9 +- 0.4^b	4.1	6.2	kidney^bliver^blung^btestis^bthymus^b	heart^ckidney^cliver^clung^cthymus^d
Females
Vehicle control	10/10	29.8 +- 0.7	13.4 +- 0.8	3.4	3.6	-	-
3900 ppm	10/10	26.1 +- 0.4^b	9.5 +- 0.4^b	3.3	5.1	-	heart^ckidney^cliver^cthymus^d
7800 ppm	10/10	21.2 +- 0.4^b	4.2 +- 0.4^b	2.3	6.4	-	heart^ckidney^cliver^clung^cthymus^c
15600 ppm	10/10	18.2 +-0.2^b	1.4 +- 0.3^b	2.3	6.5	kidney^b	heart^ckidney^cliver^clung^cthymus^c
31300 ppm	10/10	16.2 +- 0.2^b	-0.2 +- 0.2^b	2.1	5.3	kidney^bliver^blung^a	heart^ckidney^cliver^clung^cthymus^c

Significant changes in comparison to vehicle control:

^a significant decrease, p=0.05; ^b significant decrease, p=0.01; ^c significant increase, p=0.01; ^d significant increase, p=0.05

e The relevance of observed organ weight changes is discussed in "Details on results"

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the LOAELs of Citral were set at 745 and 790 mg/kg bw/day (3900ppm) in males and females respectively (based on decreased body weight).

Executive summary:

The substance Citral (Reaction mass of (E)-3,7-dimethylocta-2,6-dienal and (Z)-3,7-dimethylocta-2,6-dienal) has been tested in a study similar to OECD TG 453. Groups of male and female B6C3 mice were exposed to microencapsulated citral (greater than 96% pure) in feed for 14 weeks. The concentration of Citral in the diet was equivalent to average daily doses of approximately 745 - 8,110 mg/kg to males and 790 -  7,550 mg/kg to females.

Effects observed:In the second week of the study, four males in the high dose group were killed moribund. Lower mean body weights. Mice in the mid and high dose groups were generally thin and lethargic. The incidences of ovarian atrophy were significantly increased in females exposed to mid and high doses.

The LOAELs at 14 weeks were determined at 745 and 790 mg/kg bw/day (3900ppm) in males and females respectively (based on decreased body weight).