p-mentha-1,4(8)-diene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 January - 02 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline No 422 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

other: audited draft report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

p-mentha-1,4(8)-diene

EC Number:

209-578-0

EC Name:

p-mentha-1,4(8)-diene

Cas Number:

586-62-9

Molecular formula:

C10H16

IUPAC Name:

4-isopropylidene-1-methylcyclohexene

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

Name of the test item (as cited in the study report): TERPINOLENE MONOCONSTITUENT
IUPAC Name of the test item: 4-isopropylidene-1-methylcyclohexene
Synonym: 1-methyl-4-(1-methylethylidene)-cyclohexene; p-mentha-1,4(8)-diene
Substance type: monoconstituent
Batch No.: 119444
Purity: 97.9%
Impurities (identity and concentrations): gamma terpinene (0.7%), D and L limonene (0.1%)
This composition is within the specifications of the substance identity profile agreed within the SIEF.
Colour: colourless – slightly amber
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 15 June 2012

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Source: Charles River (UK) Limited, Margate, UK.
Age at study initiation: approximately 9 weeks
Weight at study initiation: Males: 358-409 g; Females: 210-264 g
Housing: animals were housed in groups of 5 during pre-mating for all animals, 1:1 male and female and mated females individually housed during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
Diet: ground diet (Rodent PMI 5002 (Certified), BCM IPS Limited, London, UK), ad libitum
Water: mains drinking water, ad libitum
Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS:
Temperature: 21 ± 2 °C
Humidity: 55 ± 15 %
Air changes: 15/h
Photoperiod: 12 h dark and 12 h light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: 2 % corn oil and basal laboratory diet

Details on oral exposure:

DIET PREPARATION:
Rate of preparation of diet (frequency): dietary admixtures were prepared prior to the first treatment, and on a further five occasions thereafter.

Mixing appropriate amounts with (basal laboratory diet - Rodent PMI 5002): test item was initially mixed with 2 % corn oil and subsequently a small amount of basal laboratory diet was incorporated until homogeneous, in a Robot Coupe Blixer 4 at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further nineteen minutes at a constant speed, setting 1 in a Hobart H800 mixer.

Storage temperature of food: diet was stored at approximately -18 °C.

STABILITY:
Dietary admixtures were stable for a period of three weeks at approximately -18 °C.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken from three dietary admixtures and analysed for uniformity of distribution and concentration.
Results indicated that the mean prepared dietary admixture concentrations were within acceptable ranges for the purpose of this study.

Duration of treatment / exposure:

Main phase: males were dosed daily during premating and mating periods and up to 42 days; females were dosed up to 56 consecutive days (including a three week maturation phase, pairing, gestation and early lactation for females).

Toxicity phase: females were dosed daily up to 42 consecutive days.

Recovery phase: recovery phase animals were treated with the high dose or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days.

Frequency of treatment:

once a day, 7 days a week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 800, 2500 and 5000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 54.1, 154.6 and 300.8 mg/kg bw/day
Basis:
other: equivalent to mean achieved dosages

No. of animals per sex per dose:

Main phase: 10 males and 10 females/dose (except for control males and at top dose: 5 males/dose)
Toxicity phase: 5 females/dose
Recovery phase: 5 males and 5 females /dose (control and top dose)

Control animals:

other: basal laboratory diet with 2 % corn oil added

Details on study design:

Dose selection rationale: dose levels were chosen based on the results of previous toxicity study (Study No.: 41103363).

Rationale for animal assignment: animals were allocated to dose groups using a randomisation procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.

Rationale for selecting satellite groups: to study the reversibility of toxicity effects, satellite groups (high dose and control groups) included

Post-exposure recovery period in satellite groups: 14 days

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: yes
Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: yes
Time schedule: detailed clinical observations were performed on all test and control group animals before the first exposure to the test item and for main phase males, toxicity phase females and recovery animals once weekly thereafter. Observations were also performed on main phase females weekly during the pre-mating phase and then on Days 0, 6, 13 and 20 post coitum and on Days 1 and 7 of lactation. One female treated with 800 ppm did not show positive evidence of mating, however did give birth to a live litter. Observations for this female were performed weekly until littering and the post coitum days were calculated as Days 1, 8, 14 and 20. Functional performance tests were also performed in the first five main phase males per dose group and in toxicity phase females once during the final week of treatment.

BODY WEIGHT: yes
Time schedule for examinations: individual body weights were recorded on Day 1 and then weekly for main phase males and toxicity phase females until termination. For main phase females, individual body weights were recorded on Day 1 and then weekly until pairing. Mated females were weighed on Days 0, 6, 13 and 20 post coitum and on Days 1, 4 and 7 post partum. Recovery animals were weighed on Day 1 and then weekly until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded daily for each cage of adults.

FOOD EFFICIENCY:
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for main phase and recovery males prior to and after pairing, for toxicity and recovery phase females during the recovery period where applicable and for main phase females prior to pairing.

WATER CONSUMPTION: yes
Time schedule for examinations: water intake was observed daily by visual inspection of water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY/CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: day 42 for main phase males and toxicity phase females; Day 56 for recovery group animals
Animals fasted: no
How many animals: first five main phase males and the five toxicity phase females from each test and control group; all recovery group animals
- Parameters checked:
HAEMATOLOGY: Haemoglobin, Erythrocyte count (RBC), Haematocrit, Erythrocyte indices- mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), Total leucocyte count (WBC), Differential leucocyte count- neutrophils, lymphocytes, monocytes, eosinophils, basophils, Platelet count, Reticulocyte count, Prothrombin time and Activated partial thromboplastin time were measured.
BLOOD CHEMISTRY: Urea, Glucose, Total protein, Albumin, Albumin/Globulin ratio, Sodium, Potassium, Chloride, Gamma glutamyl transpeptidase, Calcium, Inorganic phosphorus, Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine, Total cholesterol, Total bilirubin and Bile acids were measured.

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: yes
Time schedule for examinations: once before the first exposure to the test item and once weekly thereafter. Functional performance tests were performed once during the final week of treatment.

Dose groups that were examined: all groups

Battery of functions tested: sensory activity / grip strength / motor activity / other: behavioural assessments

Sacrifice and pathology:

GROSS PATHOLOGY: adult main phase males and toxicity phase females were killed by intravenous overdose of a barbiturate agent followed by exsanguination on Day 43. Adult main phase females were killed by intravenous overdose of a barbiturate agent followed by exsanguination on Day 7 post partum.
Recovery group animals were killed by intravenous overdose of a barbiturate agent followed by exsanguination on Day 57.
All animals were subject to a detailed necropsy.

ORGAN WEIGHTS:
The following organs, removed from main phase males, toxicity phase females and recovery phase animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, brain, epididymides (left and right), heart, kidneys (left and right), liver, lungs, ovaries (left and right), pituitary, prostate, seminal vesicles, spleen, testes (left and right), thymus, thyroid (weighed post-fixation with parathyroid) and uterus (with cervix and oviducts).
The following organs, removed from main phase females that were killed at the end of the study, were dissected free from fat and weighed before fixation: ovaries (left and right) and uterus (weighted with cervix and oviducts).

HISTOPATHOLOGY:
Samples of the following tissues were removed from main phase males, toxicity phase females and recovery animals and preserved in buffered 10 % formalin, except where stated: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides**, eyes*, gross lesions, heart, ileum (including peyer’s patches), jejunum, kidneys, liver, lungs (with bronchi) #, lymph nodes (cervical and mesenteric), muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, sciatic nerve, seminal vesicles, skin with mammary gland, spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, thyroid/parathyroid, trachea, testes**, thymus, urinary bladder, uterus/cervix and oviducts, and vagina.

* = eyes fixed in Davidson’s fluid; ** = preserved in Bouin’s fluid then transferred to 70 % Industrial Methylated Spirits approximately 48 h later; # = lungs were inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative

Statistics:

The following parameters were subjected to statistical anlysis: quantitative functional performance data, body weight and body weight change, food consumption and water consumption, haematology, blood chemistry, absolute and body weight-relative organ weights.

Data for males and females prior to pairing and functional performance test data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No mortality was observed.
No clinical signs that were considered to be related to test item toxicity.
One toxicity phase female treated with 5000 ppm had hunched posture on Day 41 only. In the absence of any similar effects detected in the remaining treated animals this was considered to be an isolated finding of no toxicological importance. One main phase female treated with 5000 ppm, one main phase female treated with 2500 ppm and two main phase females treated with 800 ppm had fur loss during the lactation period. Observations of this nature are commonly observed during this period and are considered to be of no toxicological significance.

BODY WEIGHT AND WEIGHT GAIN:
Reduced body weight gain was evident in animals of either sex treated with 5000 ppm (-24% in males, -50% in females) and in females treated with 2500 ppm (-41%). Males treated with 2500 ppm and females treated with 800 ppm also showed a reduction in body weight gain during the first week of treatment (-22%, -28% respectively). No such effects were detected in males treated with 800 ppm.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Reduced dietary intake was evident during the first week of treatment in animals of either sex treated with 5000 ppm (-14% in males, -24% in females). It was considered to reflect an initial reluctance to eat the diet admixture due to its low palatability.
- At 5000 ppm achieved doses for males were fairly consistent and generally maintained the intended 6-fold interval between this high dose level and the low dose level. For females at 5000 ppm, mean achieved dose was lower than the intended 6-fold interval between the low and high dose levels during the first week of administration and in toxicity phase females for the remaining treatment period.
- At 2500 ppm achieved doses were slightly lower than anticipated for main phase males during the first week of dietary exposure. After this point achieved doses were fairly consistent and generally maintained the intended 3-fold interval between this intermediate dose level and the low dose level. For females at 2500 ppm achieved doses were fairly consistent and generally maintained the intended 3-fold interval between this intermediate dose level and the low dose level.
- At 800 ppm achieved doses were generally consistent throughout the treatment period.

FOOD EFFICIENCY:
Food efficiency was reduced in animals of either sex treated with 5000 ppm. Reductions were evident during Weeks 1, 2, 4 and 5 for males and during Weeks 1 to 3 for females. No such effects were detected in males treated with 800 or 2500 ppm.

WATER CONSUMPTION:
Water consumption was considered to have been unaffected by treatment.

HAEMATOLOGY:
No adverse effects of treatment were detected in the haematological parameters examined.

CLINICAL CHEMISTRY:
No adverse effects of treatment were detected in the blood chemical parameters examined.

NEUROBEHAVIOUR:
There were no treatment related effects detected in behavioural assessments, functional performance parameters and sensory reactivity assessments.

ORGAN WEIGHTS:
Main phase males treated with 5000 ppm showed an increase in liver weight both absolute and relative to terminal body weight when compared to controls. Recovery 5000 ppm males continued to show an increase in absolute and relative liver weight following fourteen days without treatment. No toxicologically significant effects were detected in any treated toxicity phase female or in main phase males treated with 800 or 2500 ppm.

GROSS PATHOLOGY:
No macroscopic findings considered to be related to test item toxicity was observed.
One toxicity phase female treated with 5000 ppm had a mottled liver at necropsy. In the absence of any associated treatment related histology correlates in females at this level, the incidental finding was considered to be of no toxicological importance. One recovery 5000 ppm male had an enlarged mandibular lymph node at necropsy whilst one recovery 5000 ppm female had reddened lungs at necropsy. In the absence of similar effects detected in animals at the end of the treatment period, the incidental findings were considered to be of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Liver: minimal to slight centrilobular hepatocellular hypertrophy was evident in main phase males treated with 2500 and 5000 ppm. The hepatocellular hypertrophy was partly reversible in severity following fourteen days without treatment however it was still at a minimal severity in all recovery males treated with 5000 ppm.
- Kidneys: minimal to marked multifocal tubular degeneration/regeneration in the renal cortical tubules was evident in main phase males from all treatment groups. Slight to marked hyaline droplets were present in the proximal convoluted tubules and minimal to moderate granular casts were also present in the tubules of the inner cortex in one male at 800 ppm, two males at 2500 ppm and four males at 5000 ppm. The focal to multifocal tubular basophilia present incidentally in some males at 0, 800 and 2500 ppm was not evident at 5000 ppm.
Recovery 5000 ppm males showed minimal to slight tubular basophilia, minimal to slight multifocal tubular degeneration/ regeneration in the renal cortical tubules and minimal to slight hyaline droplets in the proximal convoluted tubules. Minimal to moderate granular casts were also present in the tubules of the inner cortex.

HISTORICAL CONTROL DATA:
Results were compared with historical data.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 7.5.1/1: Group mean food consumption – Main and recovery phase males

Dose Level (ppm)	Day Numbers Relative to Start Date
	From:	1	8	15	36	43	50
	To:	8	15	22	43	50	57
0 Control	Mean	29.0	27.9	28.7	28.1	24.9	27.4
	N	10	10	10	10	5	5
800	Mean	29.8	28.9	27.0	28.6	.	.
	N	10	10	10	10	.	.
2500	Mean	22.7	26.9	24.6	25.9	.	.
	N	10	10	10	10	.	.
5000	Mean	24.9	25.9	25.9	26.7	32.0	27.7
	N	10	10	10	10	5	5

                                     

 Table 7.5.1/2: Group mean food consumption – Main, toxicity and recovery phase females

Dose Level (ppm)	Day Numbers Relative to Start Date
	From:	1	8	15	22	29	36	43	50
	To:	8	15	22	29	36	43	50	57
0 Control	Mean	19.3	18.8	18.4	18.6	19.4	18.8	19.1	19.7
	N	20	20	20	10	10	10	5	5
800	Mean	18.4	18.5	17.9	19.6	17.4	20.0	.	.
	N	15	15	15	5	5	5	.	.
2500	Mean	16.4	17.3	16.0	15.3	15.4	14.9	.	.
	N	15	15	15	5	5	5	.	.
5000	Mean	14.7	16.3	15.5	14.7	14.9	14.9	18.4	16.0
	N	20	20	20	10	10	10	5	5

 

Table 7.5.1/3: Group mean food consumption – Main phase females

Dose Level (ppm)	Day Numbers
		Gestation			Lactation
	From:	0	6	13	1	4
	To:	6	13	20	4	7
0 Control	Mean	21.7	23.5	25.5	32.4	49.4
	S.D.	2.8	2.9	2.9	8.4	6.2
	N	10	10	10	10	10
800	Mean	23.7**	24.8***	27.2*	31.1	50.3
	S.D.	3.5	3.8	4.0	8.1	8.8
	N	9	10	10	10	10
2500	Mean	20.1*	21.8***	24.9	31.6	45.6
	S.D.	2.7	2.4	4.0	8.9	9.5
	N	10	10	10	10	10
5000	Mean	19.6**	20.6***	22.8***	26.4*	38.1***
	S.D.	3.5	4.7	4.3	5.5	6.6
	N	10	10	10	10	10

p<0.001 ***, p<0.01 **, p<0.05 * and p≥0.05 (not significant)

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the No-Observed-Adverse–Effect-Level (NOAEL) of terpinolene monoconstituent for systemic toxicity for females and males was 2500 and 5000 ppm, respectively (equivalent to 161.5 and 294.6 mg/kg bw/day, respectively). A combined NOAEL for males and females was determined as 154.6 mg/kg bw/day and was used for risk assessment.
Therefore terpinolene monoconstituent is not classified for repeated dose toxicity according to Directive 67/548/EEC and CLP Regulation (EC) No 1272 /2008.

Executive summary:

In a combined repeated dose toxicity study with a reproduction / developmental toxicity screening test conducted according to OECD Guideline No 422 and in compliance with GLP, three groups of Sprague-Dawley Crl: CD^®BR strain rats, each comprising of ten males and ten females for the main phase (except for control and top dose: 5 males/dose), five females for the toxicity phase and 5 males and 5 females/dose (control and top dose) for the recovery phase received terpinolene monoconstituent at dietary concentrations of 0, 800, 2500 and 5000 ppm (initially mixed with 2% corn oil). Main phase males and toxicity phase females were dosed daily 42 days. Recovery phase animals were treated with the high dose or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption, haematology, blood chemistry, oestrous cycle, organ weight, macroscopic and microscopic pathology investigations.

 

No mortality and no clinical signs related to treatment were observed. There were no treatment related effects detected in behavioural assessments, functional performance parameters and sensory reactivity assessments.

Incidences of reductions in body weight gain were evident in these animals during the treatment period resulting in an overall reduction in body weight gain. Main phase females treated with 800 ppm showed a reduction in body weight gain only during the first week of treatment. Food consumption was adversely affected at 5000 ppm in main phase animals of either sex and in toxicity and recovery phase females during the first week of treatment. It was considered to reflect a reluctance to eat the diet admixture due to its low palatability. Toxicity and recovery phase females also showed a reduction in dietary intake during the treatment period and main phase females treated with 2500 ppm. Water consumption was considered to have been unaffected by treatment.

Main phase males treated with 5000 ppm showed an increase in liver weight both absolute and relative to terminal body weight when compared to controls. Recovery 5000 ppm males continued to show an increase in absolute and relative liver weight following fourteen days without treatment. No toxicologically significant effects were detected in any treated toxicity phase female or in main phase males treated with 800 or 2500 ppm. No macroscopic findings considered to be related to test item toxicity was observed. In liver, minimal to slight centrilobular hepatocellular hypertrophy was evident in main phase males treated with 2500 and 5000 ppm. The hepatocellular hypertrophy was partly reversible in severity following fourteen days without treatment however it was still at a minimal severity in all recovery males treated with 5000 ppm. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature and does not represent an adverse health effect.

Microscopic examination also revealed effects in the kidneys of males from all treatment groups. Minimal to marked multifocal tubular degeneration/regeneration was present in males from all treated groups. These tubular findings were also accompanied by the presence of slight to marked hyaline droplets in the proximal convoluted tubules and minimal to moderate granular casts in the tubules of the inner cortex. Recovery 5000 ppm males showed minimal to slight multifocal tubular degeneration/regeneration in the renal cortical tubules and minimal to slight hyaline droplets were present in the proximal convoluted tubules. Minimal to moderate granular casts were also present in the tubules of the inner cortex. This finding is commonly observed in male rats following treatment with some xenobiotics and is not predictive of any adverse effect in humans.

No adverse effects of treatment were detected in the haematological and blood chemistry parameters examined.

Under the test conditions, the No-Observed-Adverse-Effect-Level (NOAEL) of terpinolene monoconstituent for systemic toxicity for females and males was 2500 and 5000 ppm, respectively (equivalent to 161.5 and 294.6 mg/kg bw/day, respectively). A combined NOAEL for males and females was determined as 154.6 mg/kg bw/day and was used for risk assessment.

Therefore terpinolene monoconstituent is not classified for repeated dose toxicity according to Directive 67/548/EEC and CLP Regulation (EC) No 1272 /2008.