Hydrocarbons, terpene processing by-products

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Study period:

30 March - 25 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 422 with only minor deviation: relative humidity in the experimental room transiently exceeded the target range at 2 occasions

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:

yes

Remarks:

relative humidity in the experimental room transiently exceeded the target range at 2 occasions

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:

Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

IUPAC Name:

Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Terpinolene multiconstituent
- Physical state: Clear colourless liquid
- Analytical purity: 65.6 % (sum of the main constituents >10 %)
- Composition of test material (%): Terpineols (5.6 %), alpha pinene (0.7 %), alpha fenchene (0.3 %), camphene (1.2 %), alpha phellandrene (0.5 %), alpha terpinene (2.8 %), cineol 1.4 (21.1 %), d-limonene (9.2 %), l-limonene (9.2 %), beta phellandrene (0.2 %), paracymene (0.6 %), cineol 1.8 (14.4 %), gamma terpinene (3.3%), terpinolene (30.1 %) and others (0.8 %)
- Lot/batch No.: 119445
- Date of manufacturing: 18 May 2011
- Date of receipt: 09 November 2011
- Expiration date of the lot/batch: 17 November 2012
- Storage condition of test material: 4 ºC in the dark under nitrogen

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: 396-453 g; Females: 237-293 g
- Housing: Animals were housed in groups of 5 during pre-mating for all animals, 1:1 male and female during mating 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), Harlan Laboratories U.K. Ltd., Oxon, 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 / 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 treatment, and every three weeks thereafter.
- Mixing appropriate amounts with (Type of food): Test item was initially mixed with 2 % corn oil and subsequently a small amount of basal laboratory diet was incorporated until homogeneous at a constant speed, in a Robot Coupe Blixer 4. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 mixer.
- Storage temperature of food: Diet was stored at room temperature.

STABILITY:
- Dietary admixtures were stable for three weeks at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples were taken from the 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 63 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, 2000 and 7500 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 48.9, 119.6 and 435.8 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 males from control and top dose groups: 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.: 41103360).
- 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. 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 (if feeding study):
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded on Days 0-6, 6-13 and 13-20 post coitum. For females with live litters, food consumption was recorded on Days 1, 4 and 7 post partum. Weekly food consumptions were performed for each cage of toxicity phase females and recovery group females throughout the study period. Weekly food consumptions for recovery group males were performed during the pre-pairing period, after the mating phase and during the recovery period.

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. 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 measured daily for the first three weeks of the treatment period. As there was no obvious effect of treatment on water intake during this time, no further formal gravimetric measurement of water consumption was performed for the remainder of the study, although a daily visual inspection of water bottles was performed.

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
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- 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. For all main phase females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded.

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, ovaries (left and right), pituitary (post fixation), 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).

HISTOPATHOLOGY:
- Samples of the following tissues were removed from 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 (mandibular and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, thyroid/parathyroid, trachea, testes**, thymus, urinary bladder, uterus/cervix and vagina.

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

Other examinations:

ESTROUS CYCLICITY
- 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).

Statistics:

- Body weight, food consumption during gestation and lactation, 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).

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 clinical signs that were considered to be related to test item toxicity.
- No mortality was observed.

BODY WEIGHT AND WEIGHT GAIN
- At 7500 ppm, lower mean body weight gain for males, compared to control, and mean body weight loss for females was apparent during the first week of treatment. For males, subsequent body weight gain was generally similar to control but, for body weight gain for females during the second week of treatment was still lower than control. Body weight gain of females was also lower than control during Days 1-4 of lactation.
- There was no effect of treatment on body weight at 800 and 2000 ppm throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- At 7500 ppm, mean food consumption for both sexes was lower than control during the first week of the study and was considered to reflect an initial reluctance to eat the diet admixture due to its low palatability.
- There was no effect of treatment on food consumption at 800 and 2000 ppm throughout the study.
- At 7500 ppm, achieved intake of test item for females was slightly lower for the first week of treatment compared to subsequent achieved intake for the remainder of the study. As anticipated, this resulted in the achieved dosage for this initial week being slightly lower than the intended 3.75 fold interval between the intermediate dosage and this high dosage. However, for the remainder of the study (including two weeks of pre-pairing, gestation and lactation for the main phase females) the intended difference between these dosages levels was maintained.
- For males at 7500 ppm, achieved dosage was fairly consistent throughout the study and generally maintained the intended 3.75 fold interval between the intermediate dosage and this high dosage.
- At 800 and 2000 ppm, achieved intakes of test item for both sexes were as expected and generally maintained the intended 2.5 fold interval between these dosage groups throughout the study, including for main phase females during gestation and lactation.

FOOD EFFICIENCY
- At 7500 ppm, notably inferior food conversion efficiency was apparent during the first week of the study, compared to control, for both sexes and was considered to reflect the much lower food intake for both sexes due to the low palatability of the dietary admixture.
- There was no effect of treatment on food utilisation at 800 and 2000 ppm throughout the study.

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
- At all dietary levels, main phase males showed a slight increase in absolute and body weight relative liver weights, compared to controls. At the lower dietary levels of 800 and 2000 ppm there was no dosage relationship and all individual body weight relative values were within the historical control range. This increase in liver weights was associated with adaptive liver changes during histopathological examination at all dose levels therefore it was considered as an adaptive response to the treatment.

GROSS PATHOLOGY
- No macroscopic findings considered to be related to test item toxicity was observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Liver: Centrilobular hepatocellular hypertrophy was observed in males with an incidence and/or severity proportional to the dose administered at all dietary inclusion levels. Liver of females receiving 7500 ppm also showed minimal centrilobular hypertrophy. After fourteen days of recovery, liver morphology was considered to have returned to normal. The hepatocellular hypertrophy observed was considered as an adaptive metabolic response of the liver to the presence of a xenobiotic.
- Thyroid: For males at 7500 ppm, a higher incidence of hypertrophy of the follicular epithelium was observed and considered secondary to the liver hypertrophy. One female at 7500 ppm also showed minimal hypertrophy of the epithelium. After fourteen days of recovery, thyroid morphology was considered to have returned to normal.
- Kidneys: Treatment-related lesions characterized by tubular degeneration and regeneration, granular casts, interstitial fibrosis and mixed cell infiltration, mainly of the proximal portion of the nephrons, were observed in males receiving 7500 ppm. These kidney findings were partially reversible in recovery males at 7500 ppm following the fourteen days treatment-free period, although three males still showed minimal or slight tubular degeneration, regeneration, interstitial fibrosis and mixed cell infiltration. The lesions were suggestive of alpha2μ-globulin nephropathy. It is assumed that this effect has no toxicological relevance for humans although it is considered adverse for the male rats.
- Spleen: Increased hemopoiesis was observed in two males receiving 7500 ppm and minimally in one male at 2000 ppm. Reversibility was apparent after fourteen days of recovery, with only one male at 7500 ppm showing minimal increased hemopoiesis. Because of the minimal magnitude of this finding, it was not considered an adverse effect.

HISTORICAL CONTROL DATA
- Results were compared with historical data.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

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 body weight gains – Main and recovery phases

Group (male) ppm	Increase in Body Weight (g)
	Week numbers Relative to Start Date									Week numbers Relative to Start Date
										Abs gain	Abs gain	Abs gain
	From:	1	2	3	4	5	6	7	8	1	2	7
	To:	2	3	4	5	6	7	8	9	7	7	9
Control	Mean	27.2	23.0	18.6	4.5	18.5	16.8	11.0	8.6	108.6	81.4	19.6
	S.D.	8.9	7.1	5.6	6.6	6.8	8.2	9.1	2.5	19.5	12.2	10.4
	N	10	10	10	10	10	10	5	5	10	10	5
800	Mean	22.0	16.2*	17.8	12.5	14.6	10.5	-	-	93.6	71.6	-
	S.D.	5.4	5.9	9.5	4.6	6.1	6.3	-	-	23.4	19.3	-
	N	10	10	10	10	10	10	-	-	10	10	-
2000	Mean	22.7	24.8	14.7	7.0	16.0	14.0	-	-	99.2	76.5	-
	S.D.	4.9	3.0	6.4	7.5	6.2	6.4	-	-	12.9	13.4	-
	N	10	10	10	10	10	10	-	-	10	10	-
7500	Mean	15.8**	19.3	17.3	5.6	17.8	13.1	19.8	5.0	88.1	73.1	24.8
	S.D.	7.8	5.3	5.4	9.5	8.0	6.7	11.9	6.1	13.9	15.8	15.4
	N	10	10	10	10	10	10	5	5	10	10	5

 

Table 7.5.1/2: Group mean body weight gains – Main, toxicity and recovery phases

Group (female) ppm	Increase in Body Weight (g)
	Week numbers Relative to Start Date									Week numbers Relative to Start Date
										Abs gain	Abs gain	Abs gain
	From:	1	2	3	4	5	6	7	8	1	2	7
	To:	2	3	4	5	6	7	8	9	7	7	9
Control	Mean	6.9	9.5	2.9	8.1	3.3	5.5	5.0	0.6	36.2	29.3	5.6
	S.D.	6.0	6.9	6.9	4.6	4.3	6.3	6.0	8.0	12.5	8.7	4.0
	N	20	20	20	10	10	10	5	5	20/10	20/10	5
800	Mean	4.9	5.3*	1.6	9.0	6.0	-3.6*	-	-	2.2	18.3	-
	S.D.	5.4	7.1	6.4	9.5	8.4	7.7	-	-	17.2	11.4	-
	N	15	15	15	5	5	5	-	-	15/5	15/5	-
2000	Mean	5.9	3.5*	0.7	7.4	9.2	-5.8*	-	-	20.9	15.0	-
	S.D.	6.4	6.8	6.9	7.7	3.6	5.2	-	-	14.3	9.8	-
	N	15	15	15	5	5	5	-	-	15/5	15/5	-
7500	Mean	-3.4**	4.3*	1.5	6.5	0.5	3.3	10.8	-3.2	12.7	16.1	7.6
	S.D.	6.1	5.5	5.6	4.4	5.0	6.6	4.4	4.1	9.8	9.1	7.3
	N	20	20	20	10	10	10	5	5	20/10	20/10	5

 

Table 7.5.1/3: Group mean body weight gains – Main phase

Group (female) 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
Control	Mean	31.3	32.5	93.6	17	23.2	63.8	157.4
	S.D.	10.4	4	12.5	10.8	11.6	10.7	19.9
	N	10	10	10	10	10	10	10
800	Mean	29.5	28.9	82.2	12	23.3	58.4	140.6
	S.D.	6.1	4.8	11.5	12.2	8.1	7.6	13.2
	N	10	10	10	10	10	10	10
2000	Mean	29.6	27	84	14.7	17.1	56.6	140.6
	S.D.	7.5	7.6	11	10.8	13.4	12.1	14.9
	N	10	10	10	10	10	10	10
7500	Mean	30.7	33.7	85.7	5.2	18.7	64.4	150.1
	S.D.	7.4	10	12	7.8	10.2	13.1	21
	N	10	10	10	9	9	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 findings in this study, the No-Observed-Adverse–Effect-Level (NOAEL) of Terpinolene multiconstituent for systemic toxicity for both males and females was 7500 ppm (excluding the male rat-specific effects related to alpha2μ-globulin nephropathy), equivalent to mean achieved dosage of 435.8 mg/kg bw/day.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, three groups of Sprague-Dawley Crl: CD^®BR strain rats, each comprising of ten male and ten females for the main phase (except for control and top dose: 5 males/dose), five females for the toxicity phase and 5 male and 5 females/dose (control and top dose) for the recovery phase received Terpinolene multiconstituent at doses of 800, 2000 and 7500 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 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 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, 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. At 7500 ppm, treatment was associated with lower food consumption during the first week of treatment with a concomitant reduction in mean body weight gain, or mean body weight loss in all animals. The lower food intake had been anticipated and was considered to be due to an initial reluctance to eat the low palatable treated diet. Although lower body weight gain was apparent for females during the second week of treatment, in general subsequent food intake and body weight were not adversely affected by treatment. There was no effect of treatment on body weight gain, food consumption and food utilisation at 800 and 2000 ppm throughout the study. 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. No treatment related effects were detected in mating performance, fertility and length of gestation between control and treated groups. At all dietary levels, main phase males showed a slight increase in absolute and body weight relative liver weights, compared to controls. At the lower dietary levels of 800 and 2000 ppm there was no dosage relationship and all individual body weight relative values were within the historical control range. This increase in liver weights was associated with adaptive liver changes during histopathological examination at all dose levels therefore it was considered as an adaptive response to the treatment. No macroscopic findings considered to be related to test item toxicity was observed. Centrilobular hepatocellular hypertrophy was observed in males with an incidence and/or severity proportional to the dose administered at all dietary inclusion levels. Liver of females receiving 7500 ppm also showed minimal centrilobular hypertrophy. After fourteen days of recovery, liver morphology was considered to have returned to normal. The hepatocellular hypertrophy observed was considered as an adaptive metabolic response of the liver to the presence of a xenobiotic. In thyroid, a higher incidence of hypertrophy of the follicular epithelium was observed in males at 7500 ppm and it was considered secondary to the liver hypertrophy. One female at 7500 ppm also showed minimal hypertrophy of the epithelium. After fourteen days of recovery, thyroid morphology was considered to have returned to normal. In kidneys, treatment-related lesions characterized by tubular degeneration and regeneration, granular casts, interstitial fibrosis and mixed cell infiltration, mainly of the proximal portion of the nephrons, were observed in males receiving 7500 ppm. These kidney findings were partially reversible in recovery males at 7500 ppm following the fourteen days treatment-free period, although three males still showed minimal or slight tubular degeneration, regeneration, interstitial fibrosis and mixed cell infiltration. The lesions were suggestive of alpha 2 μ-globulin nephropathy. It is assumed that this effect has no toxicological relevance for humans although it is considered adverse for the male rats. In spleen, increased hemopoiesis was observed in two males receiving 7500 ppm and minimally in one male at 2000 ppm. Reversibility was apparent after fourteen days of recovery, with only one male at 7500 showing minimal increased hemopoiesis. Because of the minimal magnitude of this finding, it was not considered an adverse effect.

Based on the findings in this study, the No-Observed-Adverse–Effect-Level (NOAEL) of Terpinolene multiconstituent for systemic toxicity for both males and females was 7500 ppm (excluding the male rat-specific effects related to alpha2μ-globulin nephropathy), equivalent to mean achieved dosage of 435.8 mg/kg bw/day.