Reaction mass of (4R)-isopropenyl-1-methylcyclohexene and (4S)-isopropenyl-1-methylcyclohexene and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 4-isopropylidene-1-methylcyclohexene

Endpoint summary

Description of key information

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, the NOAEL was 5000 ppm, equivalent to 298 mg/kg bw/day for males rats, 316 mg/kg bw/day for unmated females rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June to 29 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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)

Remarks:

November 2015

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of F0 animals at study initiation: Approximately 10 weeks
- Weight at study initiation: Males: 338-385 g; Females: 225-274 g
- Housing: No. of animals per cage - Males: pre pairing and post pairing - up to 5 animals; Females: pre
pairing - up to 5 animals; During pairing - one male and one female; Gestation - one female; Lactation:
one female + litter. Toxicity phase and recovery phase - up to five animals of one sex. Cages comprised
of a polycarbonate body with a stainless steel mesh lid. Solid (polycarbonate) bottom cages were used
during the acclimatisation, gestation, littering and lactation and maturation periods. Grid bottomed cages
were used during pairing.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

Route of administration:

oral: feed

Vehicle:

other: carrier diet, including corn oil

Details on oral exposure:

- Carrier diet: SDS VRF1 certified diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: On each occasion of the preparation of the premix the required amount of test substance and corn oil were combined and stirred. The mixture was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved or the mixture appeared dry. This mixture was then ground using a mechanical grinder. The weight of the mixture was then made up to the final weight of the premix with plain diet. The mixture was then mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of this premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a
Turbula mixer.
For Control diet, the corn oil was added directly to the diet and then prepared as indicated for the premix.
- Frequency of preparation: Weekly
- Storage of preparation: Frozen (nominally -20 °C), until required for feeding
GROUPS
Groups 1, 2, 3 and 4: 0, 800, 2500 and 5000 ppm, respectively

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Homogeneity and stability of the test material in the carrier diet was determined from Study No. OAD0009. In that study the stability that was achieved over the range 500 to
20000 ppm was: One day ambient in open containers; Eight days ambient in closed containers; 22 days frozen
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 9 of formulation were analysed for achieved concentration of the test substance.
Samples that were taken in Week 6 of treatment were discarded without analysis due to incorrect storage.

Duration of treatment / exposure:

Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of six consecutive weeks.
Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14-day period without treatment

Frequency of treatment:

Continuously (fresh diet given daily). During the recovery period, all animals were given untreated diet (without corn oil).

Dose / conc.:

800 ppm

Remarks:

nominal in diet

Dose / conc.:

2 500 ppm

Remarks:

nominal in diet

Dose / conc.:

5 000 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

Toxicity phase : 5 males/dose for vehicle and 5000 ppm dose groups; 10 males/dose for 800 and 2500 ppm dose groups
Reproductive phase: 10 females/dose
Recovery phase: 5 animals/sex/dose for vehicle and 5000 ppm dose groups

Control animals:

other: control group was provided with the carrier diet, including corn oil

Details on study design:

- Dose selection rationale: Dose levels were selected in conjunction with the Sponsor following the completion of the dose range finder study (Study number OAD0019). In the dose range finding study the
doses of 1500, 7500 or 15000 ppm were well tolerated, there were no mortalities and no adverse clinical signs or macroscopic changes detected. The body weght effect observed in males and females receiving 15000 ppm was considered too marked for use in an OECD 422 study. The magnitude of body weight
effect at 7500 ppm for females was also considered to preclude this dose for use during gestation, parturition and early lactation.
- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately

Positive control:

Not applicable

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each adult animal. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery phase animals in Group 1 and 4 and the five lowest numbered toxicity males in Group 2 and 3 during Week 6 of treatment.
- Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3.

BODY WEIGHT: Yes
- Time schedule for examinations:
Toxicity phase males and recovery phase males and females: Before feeding on the day that treatment commenced (Day 1), weekly thereafter and before necropsy.

- Time schedule for examinations:
Toxicity phase males, recovery phase males and females and reproductive phase females before pairing: Daily. From these records the mean daily consumption per animal (g/rat/day) was calculated for each cage; Food consumption was not recorded for males during the period when paired for mating (Day 22 to
26). Food consumption recordings recommenced on Day 27.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematology: Week 3 (prior to pairing) - Five lowest numbered reproductive phase females per group and recovery phase females; Week 6 - Five lowest numbered toxicity phase males per group; Recovery Week 2 - All surviving recovery phase animals
Clinical chemistry: Week 3 (prior to pairing) - Five lowest numbered reproductive phase females per group and recovery phase females; Week 6 - Five lowest numbered toxicity phase males per group
- Animals fasted: Yes, Blood samples were collected after overnight withdrawal of food.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte co
unt: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count,
Morphology: Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

Sacrifice and pathology:

SACRIFICE
- Toxicity phase animals: Following completion of Week 6 investigations.
- F0 females: Scheduled kill - Day 7 of lactation; Failing to produce viable litter - Day 25 after mating.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together.
Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
The five lowest numbered surviving toxicity phase males and the five lowest numbered reproductive phase females with live litter in Groups 1 and 4 at scheduled termination
Abnormalities only: All animals
Kidneys: All males from the toxicity (Group 2 and 3) and recovery (Group 1 and 4) phases which had tissues retained.
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid Schiff (PAS) method.
- Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes in modified Davidson’s fluid; Eyes In Davidson’s fluid

Statistics:

See section "Any other information on materials and methods incl. tables”.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence (7/15 compared to 0/15 in Control) of vocalisation was apparent for females receiving 5000 ppm; this is not an uncommon finding in this species but may indicate that females at
this dietary concentration were more irritable. Brown staining affected the head or upper dorsal thorax of animals during the study, males and females were still affected during the recovery phase. These
observations lacked a dose relationship indicating it is unlikely to be related to the administration of Multiconstituent Dipentene

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male that previously received 5000 ppm (Group 4 Male animal number 40) was found dead on Day 8 of recovery. None of the macroscopic or microscopic findings were considered to be related to treatment or have contributed to the death of this animal; the cause of death was undetermined. In the absence of any other deaths in this dose group an association to treatment is considered unlikely

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain up to pairing (Days 1 to 22) and for the complete treatment period was low among animals receiving 2500 or 5000 ppm although statistical significance (p<0.05) was only attained for recovery phase females receiving 5000 ppm during Days 1 to 43; a dose dependant trend was not apparent for females. During recovery animals that had previously received 5000 ppm displayed statistically higher body weight gain when compared with Controls (p<0.01 for males and p<0.05 for females).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Weekly mean food consumption data indicated males receiving 2500 or 5000 ppm throughout the dosing period and all females receiving 5000 ppm before pairing was marginally lower than Controls this difference was not considered to be adverse at the degree observed. The food consumption during recovery was noted to be slightly higher than Control for animals that previously received 5000 ppm.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

During Week 6 of study, males receiving 5000 ppm were recorded to be slightly but statistically significantly low haematocrit, haemoglobin concentration and large unstained cell count. However these data
were within historical control data. During recovery these parameters for males which had previously received 5000 ppm were similar to Control.

During Week 3 of study, females receiving 5000 ppm indicated slightly low reticulocytes, mean cell haemoglobin concentration, red cell distribution width and slightly increased mean cell volume all differences attaining statistical significance. However these data are within historical control data. After 14 days recovery there were no statistical differences to the Control.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related to treatment with Multiconstituent Dipentene were observed in the male kidney. Minimal hyaline droplets within the cortical tubules were observed in all kidneys examined microscopically from males that received 5000 or 2500 ppm and the majority of males that received 800 ppm.
A similar change was not observed in the kidneys of any females.
Microscopically there was minimal hyaline droplet accumulation in the cortical tubules of the kidneys of males only at all dose levels and this is consistent with the accumulation of alpha 2u globulin. This common pathological change has been associated with the exposure to a variety of hydrocarbons-containing chemicals, and is restricted to male rats. The minimal levels of droplet accumulation observed were considered to be non-adverse. In addition, following a 2-week recovery period hyaline droplets were not observed in the kidneys of any males previously receiving 5000 ppm, indicating complete recovery. No pathological changes observed in the reproductive tissues examined were considered to be related to the administration of Multiconstituent Dipentene. This observation is not relevant to humans.
A similar change was not observed in the kidneys of any females.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
- No adverse signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to treatment.
- An increased incidence (7/15 compared to 0/15 in Control) of vocalisation was apparent for females receiving 5000 ppm; this is not an uncommon finding in this species but may indicate that females at this dietary concentration were more irritable. Brown staining affected the head or upper dorsal thorax of
animals during the study, males and females were still affected during the recovery phase. These observations lacked a dose relationship indicating it is unlikely to be related to the administration of test item.
- On Day 8 of recovery one male that previously received 5000 ppm was found dead. The cause of death was undetermined but considered of unlikely relationship to treatment.

BODY WEIGHT
- Body weight gain up to pairing (Days 1 to 22) and for the complete treatment period was low among animals receiving 2500 or 5000 ppm although statistical significance (p<0.05) was only attained for recovery phase females receiving 5000 ppm during Days 1 to 43; a dose dependant trend was not apparent
for females. During recovery animals that had previously received 5000 ppm displayed statistically higher body weight gain when compared with Controls (p<0.01 for males and p<0.05 for females).

FOOD CONSUMPTION
- Weekly mean food consumption data indicated males receiving 2500 or 5000 ppm throughout the dosing period and all females receiving 5000 ppm before pairing was marginally lower than Controls this difference was not considered to be adverse at the degree observed. The food consumption during
recovery was noted to be generally slightly higher than Control for animals that previously received 5000 ppm.

TEST SUBSTANCE INTAKE:
- Overall mean achieved doses at 800, 2500 or 5000 ppm were 50, 148 or 298 mg/kg bw/day for males, respectively. Overall mean achieved dose levels for recovery females throughout receiving 800, 2500 or 5000 ppm were 53, 174 or 316 mg/kg bw/day, respectively.

ORGAN WEIGHTS
- Organ weights for toxicity phase males killed after 6 weeks of treatment, and recovery males and females killed following 2 weeks of recovery indicated no adverse effects of dosing.
- Males at 5000 ppm terminated after Week 6 of study had slightly low adjusted mean prostate and seminal vesicle weights (0.85X and 0.86X Control, respectively) but these differences did not attain statistical significance. At the end of the 2-week recovery period prostate and seminal vesicle weights were similar
to Controls for males which had previously received 5000 ppm.

GROSS PATHOLOGY:
- Examination of males after 6 weeks of dosing and males and females following a 2 week recovery period did not reveal any findings that could be attributed to treatment.

HISTOPATHOLOGY:
Males killed after 6 weeks of dosing: Treatment related findings: Minimal hyaline droplets within the cortical tubules were observed in all kidneys examined microscopically from males that received 5000 or 2500 ppm and the majority of males that received 800 ppm. A similar change was not observed in the kidneys of any females.
Incidental findings: All other findings were considered to be incidental and unrelated to treatment and were consistent with common background microscopic changes previously seen in control Sprague Dawley rats.
Animals killed after 2 weeks of recovery
Treatment related findings: The kidneys from all recovery males and any abnormalities identified at the macroscopic examination were examined microscopically. No findings were considered to be related to treatment.
The incidence and distribution of all findings were consistent with common background changes previously seen in control Sprague Dawley rats.
OTHER FINDINGS:
OTHER FINDINGS (PARENTAL ANIMALS)
- Sensory reactivity and grip strength: Sensory reactivity and grip strength examined during Week 6 of dosing and Days 4 to 6 of lactation (females only) were considered unaffected by the administration of test item.
- Motor activity: There was no effect on either rearing (high beam) or ambulatory (low beam) motor activity.
- Haematology: Analysis of haematological parameters during Week 3 of study for females and Week 6 of study for males revealed low haematocrit and haemoglobin and large unstained cell counts, in males given 5000 ppm. Females receiving 5000 ppm had low reticulocytes (absolute count and percentage), mean cell haemoglobin concentration, and red cell distribution width and a slight increase in mean cell volume.
There were no statistical differences to the Control 14 days after the cessation of dosing.
- Biochemistry: Analysis of biochemical parameters during Week 6 of study for males and Week 3 of study for males indicated there were no adverse effects of the administration of test item. In males, a statistical significance was attained for low alanine amino transferase activity at all dose levels (0.81X to 0.84X Control), a dose response was not apparent and a change of this degree was considered not to be adverse. Females receiving 2500 or 5000 ppm showed slightly low alanine amino transferase activity (0.78X Control for both), with 5000 ppm attaining statistical significance and females at 5000
ppm showed significantly low cholesterol levels (0.90X Control). The degree of change recorded was considered not to be adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed at the highest concentration tested

Dose descriptor:

NOAEL

Effect level:

298 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effect observed at the highest concentration tested

Dose descriptor:

NOAEL

Effect level:

316 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effect observed at the highest concentration tested

Critical effects observed:

no

Lowest effective dose / conc.:

2 500 ppm

System:

urinary

Organ:

kidney

Formulation analysis:

The mean concentrations of test item in test formulations analysed for the Week 1 were within applied limits of +10 %/-15 % of nominal concentrations, confirming accurate formulation. The investigation into Week 9 showed that the concentration of Alpha‑terpinene in both the formulations and procedural recoveries had

reduced by approximately 50 % due to the oxidization of the sub-sample of Multiconstituent Dipentene used for the analyses over the course of the study. When the mean Week 1 procedural recovery is used to correct the Week 9 formulations, Group 2 is outside acceptable limits with an RME of -20.6 % from the nominal concentration, Groups 3 and 4 are both within applied limits.

Table 7.8.1/4: Analysed concentrations for Multiconstituent Dipentene in SDS VRF1 certified diet:

Occasion	Group	Nominal inclusion (ppm)	Analysed concentration (ppm)			RME (%)	Difference from mean (%)	Procedural recoveries (%)
			Analysis 1	Analysis 2	Mean			At analysis	Mean
Week 1	1	0	ND	ND	-	-	-
	2	800	770	772	771	-3.6	±0.16	87.9	87.2
	3	2500	2400	2480	2390	-4.4	±0.43	86.0
	4	5000	4950	5220	5090	+1.8	±2.62	87.8
Week 9	1	0	ND	ND	-	-	-
	2	800	632	637	635	-20.6	±0.40	-	87.2^1
	3	2500	2220	2240	2230	-10.8	±0.53	-
	4	5000	4370	4450	4410	-11.8	±0.85	-

RME: Relative mean error, representing the deviation from nominal

ND: Not detected

¹ Week 9 results are corrected for the mean of Week 1 procedural recoveries

Conclusions:

The NOAEL for this screening study was 5000 ppm, equivalent to 298 mg/kg bw/day for males, 316 mg/kg bw/day for unmated females.

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, groups of Crl:CD(SD) rats received REACTION MASS OF DL-LIMONENE, ALPHA‑ GAMMA- TERPINENES, TERPINOLENE orally, via the diet, at concentrations of 800, 2500 or 5000 ppm.

Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of six consecutive weeks. Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14-day period without treatment. Recovery phase males were used for pairing with reproductive phase females, but recovery females were not paired. A similarly constituted Control group was assigned to each phase, and received the vehicle, untreated diet, throughout the same relative treatment period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology, blood chemistry, oestrous cycles, organ weight and macroscopic investigations were undertaken for adult animals. Microscopicpathology investigations were undertaken for the first five toxicity phase males in Group 1 (Control) and Group 4 (5000 ppm) with examination of kidneys from males of Group 2 and 3.

Test item at dietary concentrations of 800, 2500 or 5000 ppm resulted in overall mean achieved doses of 50, 148 or 298 mg/kg bw/day for males, respectively. The overall mean achieved dose levels for unmated females were 53, 174 or 316 mg/kg bw/day.

On Day 8 of recovery one male that previously received 5000 ppm was found dead. The cause of death was undetermined but considered of unlikely relationship to treatment. Test item was well tolerated, with no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, blood chemistry, organ weights and macroscopic pathology.

Overall body weight gain and weekly mean food consumption data for the dosing period were generally low for animals receiving 2500 or 5000 ppm. During recovery, animals that had previously received 5000 ppm showed statistically significantly high body weight gain coupled with a slight increase in food consumption.

During gestation body weight gain and mean food consumption for all treated females were generally similar to Control values.

Analysis of haematological parameters during Week 3 of study for females and Week 6 of study for males revealed low haematocrit and haemoglobin and large unstained cell counts, in males given 5000 ppm. Females receiving 5000 ppm had low reticulocytes (absolute count and percentage), mean cell haemoglobin concentration, and red cell distribution width and a slight increase in mean cell volume. There were no statistical differences to the Control 14 days after the cessation of dosing.

Microscopically there were minimal hyaline droplets within the cortical tubules observed for all males examined (5/5) that received 2500 or 5000 ppm and the majority of males examined (4/5) at 800 ppm. This change was only observed in males and considered to be related to the administration of test item. This change was not observed in the kidneys of any males that previously received 5000 ppm following a 2-week recovery period, indicating complete recovery.

Therefore, the NOAEL for this screening study was 5000 ppm, equivalent to 298 mg/kg bw/ day for males, 316 mg/kg bw/day for unmated females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

307 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study in accordance with OECD 422 guidelines

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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, groups of Crl:CD(SD) rats received REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE orally, via the diet, at concentrations of 800, 2500 or 5000 ppm. Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of six consecutive weeks. Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14-day period without treatment. A similarly constituted Control group was assigned to each phase, and received the vehicle, untreated diet, throughout the same relative treatment period.

Test item at dietary concentrations of 800, 2500 or 5000 ppm resulted in overall mean achieved doses of 50, 148 or 298 mg/kg bw/day for males, respectively. The overall mean achieved dose levels for unmated females were 53, 174 or 316 mg/kg bw/day.

Test item was well tolerated, with no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, blood chemistry, organ weights and macroscopic pathology.

Overall body weight gain and weekly mean food consumption data for the dosing period were generally low for animals receiving 2500 or 5000 ppm. During recovery, animals that had previously received 5000 ppm showed statistically significantly high body weight gain coupled with a slight increase in food consumption.

Analysis of haematological parameters during Week 3 of study for females and Week 6 of study for males revealed low haematocrit and haemoglobin and large unstained cell counts, in males given 5000 ppm. Females receiving 5000 ppm had low reticulocytes (absolute count and percentage), mean cell haemoglobin concentration, and red cell distribution width and a slight increase in mean cell volume. There were no statistical differences to the Control 14 days after the cessation of dosing.

 

Microscopically there were minimal hyaline droplets within the cortical tubules observed for all males examined (5/5) that received 2500 or 5000 ppm and the majority of males examined (4/5) at 800 ppm. This change was only observed in males and considered to be related to the administration of test item. This change was not observed in the kidneys of any males that previously received 5000 ppm following a 2-week recovery period, indicating complete recovery.

Therefore, the NOAEL for this screening study was 5000 ppm, equivalent to 298 mg/kg bw/ day for males, 316 mg/kg bw/day for unmated females .

Justification for classification or non-classification

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, in rats, the NOAELwas 5000 ppm (maximal tested dose), equivalent to 298 mg/kg bw/day for males, 316 mg/kg bw/day for unmated females. No toxic adverse effects, relevant for human, were observed , therefore the registered substance is not classified for repeated dose toxicity according to CLP Regulation (EC) No. 1272/2008.