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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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:

study 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 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.

Details on mating procedure:

M/F ratio per cage: 1/1
Length of cohabitation: up to 14 days
Proof of pregnancy: presence of vaginal plug and sperm in vaginal smear referred to as Day 0 of gestation.
After successful mating each pregnant female was caged individually.

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).

Frequency of treatment:

once a day, 7 days a week

Details on study schedule:

none

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 achived 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)

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.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

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 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.

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 (with the exception of the mating phase animals where applicable).
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for main phase males prior to and after pairing and for main phase females prior to pairing. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females, during gestation and lactation.


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

OTHER:

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. Gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defecation, transfer arousal and tail elevation were evaluated.

FUNCTIONAL PERFORMANCE TESTS:
Motor activity and forelimb/hindlimb grip strength were evaluated.

SENSORY REACTIVITY:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. Grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, blink reflex and startle reflex were evaluated.

HAEMATOLOGY AND BLOOD CHEMISTRY:
Haematological and blood chemical investigations were performed on the first five main phase males from each test and control group prior to termination (Day 42). In addition haematological and blood chemical investigations were performed on all recovery group animals after the fourteen day treatment-free period at termination (Day 56). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
- Haematology parameters: 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 parameters: 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.

PREGNANCY AND PARTURITION:
Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and around the expected date of parturition. Observations were carried out at approximately 08:30 and 12:30 hours at weekends and public holidays.

Oestrous cyclicity (parental animals):

Animals were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, detailed histological examination of the testes and epididymes with special emphasis on stages of spermatogenesis

Litter observations:

PARAMETERS EXAMINED:
On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

All litters were examined for number of offspring born, number of offspring alive recorded daily and reported on Days 1, 4 and 7 post partum, sex of offspring on Days 1 and 4 post partum, clinical condition of offspring from birth to Day 7 post partum and individual offspring weights on Days 1, 4 and 7 post partum.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE:
Adult main phase males 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.

GROSS NECROPSY:
All animals were subject to a detailed necropsy. For all main phase females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The following organs, removed from main phase males 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 (weighed 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).

Samples of the following tissues were removed from main phase males 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

Postmortem examinations (offspring):

SACRIFICE:
Surviving offspring were terminated via intracardiac overdose of a barbiturate agent.

GROSS NECROPSY:
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Body weight, food consumption, pre-coital interval and gestation length, litter size and weights, sex ratio, implantation sites, implantation loss and viability indices, offspring body weight and change, haematology, blood chemistry, adult absolute and body weight-relative organ weights were subjected for statistical analysis.

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).

Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test.

Non-parametric methods were used to analyse implantation loss, offspring sex ratio and landmark developmental markers. Probability values (p) were calculated as follows: p<0.001 ***, p<0.01 **, p<0.05 * and p≥0.05 (not significant).

Reproductive indices:

Mating Performance and Fertility
Pre-coital Interval: calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

Fertility Indices
Mating Index (%): No. of animals mated x 100/No. of animals paired
Pregnancy Index (%): No. of pregnant females x 100/No. of animals mated

Gestation and Parturition Data
Gestation Length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
Parturition Index (%): No. of females delivering live offspring x 100/No. of pregnant females

Offspring viability indices:

Implantation Losses (%)
Post–implantation loss: [(No. of implantation sites-Total no. of offspring born)/No. of implantation sites]x100

Live Birth and Viability Indices
Live Birth Index (%): No. of offspring alive on Day 1 x 100/No. of offspring born
Viability Index (%): No. of offspring alive on Day 4 x 100/No. of offspring alive on Day 1
Lactation index (%): No. of offspring alive on Day 7 x 100/No. of offspring alive on Day 1

Sex Ratio (% males)
Sex ratio was calculated for each litter on Days 1, 4 and 7 post partum using the following formula: No. of male offspring x 100/Total no. of offspring

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
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.
There were no treatment related effects detected in behavioural assessments, functional performance parameters and sensory reactivity assessments.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- 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%). This resulted in lower body weights for main phase females treated with 5000 ppm during gestation and lactation. Body weight gains during lactation were also reduced for these females (-71% overall). 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.
- Reduced dietary intake was evident during the first week of treatment in animals of either sex treated with 5000 ppm (-14% males, -24% females). It was considered to reflect an initial reluctance to eat the diet admixture due to its low palatability. Main phase females treated with 5000 ppm continued to show a reduced dietary intake throughout gestation and lactation. Main phase females treated with 2500 ppm showed a reduced dietary intake during the first two weeks of gestation. Food efficiency was also adversely affected at 5000 ppm. No such effects were detected in males treated with 800 or 2500 ppm.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS):
- At 5000 ppm achieved doses 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.

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

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS):
No treatment related effects were observed in estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
No treatment-related effects were detected in mating performance, fertility and gestation lengths:
- all animals mated within the first five days of pairing;
- there were no differences in conception rates for treated animals;
- the distribution of gestation lenghts for treated females was comparable to controls. The majority of females showed a gestation lenghts between 21 1/2 and 23 days.

ORGAN WEIGHTS (PARENTAL ANIMALS):
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.No toxicologically significant effects were detected in main phase males treated with 800 or 2500 ppm.

GROSS PATHOLOGY (PARENTAL ANIMALS):
No macroscopic findings considered to be related to test item toxicity was observed.

HISTOPATHOLOGY (PARENTAL ANIMALS):
- 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 (in 4 out of 5 males at 800 ppm). 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.

OTHER FINDINGS (PARENTAL ANIMALS)

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

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

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

161.5 mg/kg bw/day

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOAEL

Remarks:

(reproductive toxicity)

Effect level:

294.6 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on reproductive parameters were observed up to the highest dose tested 5000 ppm (corresponding to 294.6 and up to 371 mg/kg bw/day) for males and females (Gestation Day 0-6) respectively).

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING):
No treatment related effects were detected in litter viability for litters from treated females compared to controls.

CLINICAL SIGNS (OFFSPRING):
No clinical signs related to treatment were observed.

BODY WEIGHT (OFFSPRING):
Reduced litter weights were evident for litters from main phase females treated with 5000 ppm on Day 7 post partum when compared to controls. Mean offspring weights, both male and female were reduced from females treated with 5000 ppm on Day 7 post partum. Cumulative body weight gains for these offspring were reduced throughout lactation compared to controls.

SEX RATIO (OFFSPRING):
No treatment related effects were detected in sex ratio for litters from treated females compared to controls.

GROSS PATHOLOGY (OFFSPRING):
There were no macroscopic findings considered to be related to test item toxicity.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

(developmental toxicity)

Generation:

F1

Effect level:

356 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 7.8.1/1: Group mean body weight gains – Main phase females

Dose Level (ppm)		Increase in Body Weight (g)					Cumulative Body Weight Change (g)
		Days					Days
		Gestation			Lactation		Gestation
	From:	0	6	13	1	4	0	0
	To:	6	13	20	4	7	13	20
0 Control	Mean	24.7	31.4	88.8	15.1	16.7	56.1	144.9
	S.D.	6.2	4.9	10.1	7.7	8.1	5.0	13.7
	N	10	10	10	10	10	10	10
800	Mean	31.1	34.3	98.3	12.5	15.7	65.4	163.7
	S.D.	7.2	7.3	11.0	5.8	6.6	13.0	20.5
	N	10	10	10	10	10	10	10
2500	Mean	20.6	31.6	83.3	12.2	18.1	52.2	135.5
	S.D.	12.0	6.6	14.7	9.6	5.2	10.2	20.8
	N	10	10	10	10	10	10	10
5000	Mean	21.6	28.0	79.7	2.3**	7.0*	49.6	129.3
	S.D.	4.5	3.4	12.7	6.4	10.3	6.2	13.0
	N	10	10	10	10	10	10	10

 

Table 7.8.1/2: 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 7.8.1/3: 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 7.8.1/4: 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 (corresponding 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.
The NOAEL for reproductive toxicity was 5000 ppm (corresponding to 294.6 mg/kg bw/day) and the NOAEL for maternal toxicity and developmental toxicity was 2500 ppm (corresponding to 356 mg/kg bw/day).
Therefore terpinolene monoconstituent is not classified for reproduction 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) received terpinolene monoconstituent at doses of 800, 2500 and 5000 ppm by dietary admixture (initially mixed with 2% corn oil). Main phase males were dosed daily during premating and mating periods and up to 42 days and females were dosed up to 63 consecutive days (including a three week maturation phase, pairing, gestation and early lactation for females). 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, mating performance, fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken.

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 main phase females throughout gestation and lactation. It was considered to reflect a reluctance to eat the diet admixture due to its low palatability. Main phase females treated with 2500 ppm showed incidences of slight reduced dietary intake during gestation.

Water consumption was considered to have been unaffected by treatment.

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

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. No toxicologically significant effects were detected 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.

Reduced litter weights were evident for females treated with 5000 ppm on Day 7 post partum when compared to controls. Mean offspring weights were reduced from these litters on Day 7 post partum resulting in a reduction in body weight gain between Days 4 and 7 post partum. In the absence of any effect in litter size or litter viability the intergroup differences detected in offspring development were considered to be related to the decline in heath of the adult female rather than a direct toxic effect on the offspring.

No treatment-related effects were detected in mating performance, fertility and gestation lengths:

- all animals mated within the first five days of pairing;

- there were no differences in conception rates for treated animals;

- the distribution of gestation lenghts for treated females was comparable to controls. The majority of females showed a gestation lenghts between 21 1/2 and 23 days.

 

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 (corresponding to 161.5 and 294.6 mg/kg bw/day, respectively), the NOAEL for reproductive toxicity was 5000 ppm (corresponding to 294.6 mg/kg bw/day) and the NOAEL for maternal toxicity and developmental toxicity was 2500 ppm (corresponding to 356 mg/kg bw/day).

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