Reaction mass of 3-isopropyl-6-methylenecyclohexene and (4R)-1-methyl-4-(prop-1-en-2-yl)cyclohexene and (4S)-1-methyl-4-(prop-1-en-2-yl)cyclohexene

Endpoint summary

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD Guideline 422), the NOAEL for systemic toxicity was 1500 ppm (mean achieved doses of 85 mg/kg bw/day for males and 94 mg/kg bw/day for toxicity phase females, corresponding to a mean NOAEL of 89.5 mg/kg bw/day).

In a GLP and OECD 413 compliant 90-day repeated dose toxicity study by inhalation with the registered substance, the No Observed Adverse Effect Concentration (NOAEC) was considered to be 0.765 mg/L, based on no adverse effects observed at the highest concentration tested.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

89.5 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

GLP study conducted according to OECD guideline 422 with one dose missing (high dose group was terminated earlier due to welfare reasons).

System:

other: decrease in food consumption and bodyweight due to palatability issues

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

765 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

GLP study conducted according to OECD guideline 413

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

765 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP study conducted according to OECD guideline 413

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

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

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 the test item, Reaction mass of beta-phellandrene and D-limonene and L-limonene orally, via the diet at concentrations of 0,1500, 4000/3500/4000, 10000 ppm (Group 1, 2, 3 and 4, respectively). After 43 days of treatment all the Group 4 animals were prematurely sacrificed, due to effects seen. Toxicity phase males and females of Groups 1 to 3 were treated for at least seven weeks. The males were paired with the reproductive phase females after six weeks of treatment. Males and females were terminated in Week 8 on completion of the Week 8 investigations.Reproductive phase females were treated for six weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation.A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 certified diet with corn oil throughout the same relative period. During the study, clinical condition, detailed physical examination and arena observations, body weight, food consumption, ophthalmic examination, sensory reactivity, grip strength, motor activity, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken.

Achieved dose generally maintained the intervals between dietary concentrations. During lactation achieved doses were higher reflecting the increased physiological demand on the dams.

Two females at 4000/3500/4000 ppm and their litters were killed for welfare reasons due to poor clinical condition on Day 1 of lactation. Since both females showed poor clinical condition on Day 1 of lactation and necrosis in the liver a relationship to treatment cannot be completely ruled out. There were no clinical signs or arena observations considered to be related to treatment amongst males and females at 1500 or 4000/3500/4000 ppm.

The body weight, body weight gain and food consumption of males receiving test item at 4000/3500/4000 ppm was similar to that of the Control throughout the treatment period. Females allocated to the toxicity phase and receiving test item at 4000 ppm experienced bodyweight loss in Week 1, bodyweight stasis in Week 2 and during Weeks 3 to six bodyweight gain was similar to that of Control. The dose was lowered to 3500 ppm from Day 26, and increased to 4000 ppm from Day 40 of treatment. Overall body weight gain during Days 1-56 was 21% of Control. Females allocated to the reproductive phase and receiving test item at 4000 ppm experienced bodyweight loss in Week 1 with food consumption also lower than Control in this week, bodyweight stasis in Week 2 and during Weeks 3 to 6 bodyweight gain was similar to that of Control. Food consumption of these females was generally similar to that of the Controls from Week 2 onwards. The dose was lowered to 3500 ppm from Day 26, and increased to 4000 ppm from Day 40 of treatment. At 4000/3500/4000 ppm mean bodyweights and food consumption during gestation were lower than in Controls. Bodyweight gain during Days 0-13 of gestation was similar to Control but weight gain was low during Days 13-20 of gestation. During lactation, the females at 4000/3500/4000 ppm showed bodyweight loss during Days 1-7, compared to weight gain in the Controls, and body weight gain was similar to that of the Control from Days 7-13. Mean food consumption during lactation was slightly lower than in Controls. 

There were no findings at ophthalmic examination that were considered related to treatment with the test item. There was no effect of treatment on grip strength, motor activity and sensory reactivity.

At haematological examination in Week 8 of treatment, there were no significant changes amongst males at 4000/3500/4000 ppm or 1500 ppm. In females, white blood cell, neutrophil, lymphocyte, monocyte and large unstained cell counts were lower at 4000/3500/4000 ppm or 1500 ppm when compared with that of the Control. At blood chemistry examination in Week 8 of treatment, there were no significant changes amongst males or females at 4000/3500 ppm or 1500 ppm. There were some minor differences from Control, however, these occurred in one sex only and are considered unlikely to be related to treatment with test item. At examination of the urine in Week 8 of treatment, there were no significant changes amongst males or females at 4000/3500/4000 ppm or 1500 ppm. Protein levels but not total protein output were statistically significantly high for females in the 4000/3500/4000 ppm group, no other changes achieved statistical significance.

After 8 weeks of treatment, the mean adjusted and unadjusted liver weights among males and females at 4000/3500/4000 ppm was marginally high compared with Controls.

The macroscopic examination performed after eight weeks of treatment or reproductive phase revealed no test-item related lesions.At pathological examination, administration of test item to rats by diet for at least 5 weeks resulted in treatment related effects in the kidneys (increased incidence and/or severity of cortical tubule hyaline droplet accumulation) in males treated with 1500 or 4000/3500/4000 ppm.This finding was considered not to represent an adverse effect of treatment as the survival and clinical condition of the animals was unaffected and there was no evidence of impaired renal function (urea and creatinine levels were similar to Controls).

Based on the results of this study it is concluded that the No-observed-adverse-effect-level (NOAEL) of test item for systemic toxicity was 1500 ppm (mean achieved doses of 85.4 mg/kg bw/day for males, 93.6 mg/kg bw/day for toxicity phase females, 106.9 mg/kg bw/day for females during gestation and 176.9 mg/kg bw/day for females during lactation).

 

• 90-day repeated dose toxicity study by inhalation (OECD 413)

In a repeated dose toxicity study conducted according to OECD Guideline 413 and in compliance with GLP, Reaction mass of beta-phellandrene and d-limonene and l-limonene was administered by inhalation-aerosol to groups of Sprague Dawley rats (10 rats/sex/group) by snout-only inhalation exposure at target exposure levels of 0.10, 0.25 and 0.75 mg/L for 6 hours per day, 5 days per week for 13 weeks. Control animals received air only. Recovery animals were similarly treated for 13 weeks followed by a 4 week off dose period.

During the study, clinical condition, body weight, food consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry, organ weight, broncho-alveolar lavage examinations, macropathology and histopathology investigations were undertaken.

 

The achieved aerosol concentrations were 108, 94 and 102% of the target concentrations for Groups 2, 3 and 4, respectively, for an achieved exposure concentrations (mean of daily means) of 0.108, 0.234 or 0.765 mg/L.
The particle diameters for Groups 2, 3 and 4 indicate the generated aerosols were respirable to the rat.

Test item related clinical signs following exposure, and occasionally persisting to the end of working day, included elevated gait in a small number of males and females exposed to 0.234 mg/L and males exposed to 0.765 mg/L on one or a few occasions and in all females exposed to 0.765 mg/L on one or generally more occasions. Abnormal gait including flattened, swaying or splayed hind limbs was seen in a small number of females exposed to 0.765 mg/L on a few occasions. Red staining on the coat was noted at the weekly examination in some males and females exposed to 0.234 and 0.765 mg/L and some females exposed to 0.108 mg/L, with the highest number of animals affected being exposed to 0.765 mg/L.

Lower body weight gain over the 13 weeks of exposure in males and females exposed to 0.765 mg/L and females exposed to 0.234 mg/L and slightly lower food consumption over the same period in animals exposed to 0.765 mg/L was seen. Full recovery was seen by the end of the recovery phase.

There were no test item-related effects on ophthalmoscopic changes, or on haematology, blood chemistry or urinalysis parameters investigated in Week 13 or in broncho-alveolar lavage responses at termination.

There were no test-item related effects on organ weights or the incidence of macroscopic or microscopic pathology findings. The incidence of testicular tubular degeneration/atrophy correlating with lower testes weight, in males exposed to 0.765 mg/L was considered of uncertain relationship to treatment with the test item and, due to the small numbers affected and the absence of findings in the recovery animals, not considered adverse.  

 

Therefore, exposure at 0.234 or 0.765 mg/L was associated with non adverse effects including abnormal but transient signs on occasion and lower body weight gain and/or food consumption which showed full recovery. 

There were no test item-related pathological findings indicative of systemic or local toxicity, although a low incidence of testicular tubular degeneration/atrophy was not considered adverse.

In conclusion, the No Observed Adverse Effect Concentration (NOAEC) was considered to be 0.765 mg/L, based on no adverse effects or test item-related pathological findings observed at the highest concentration level tested.

 

Justification for classification or non-classification

Harmonised classification:

The test material has no harmonised classification for repeated dose toxicity according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

The NOAEL obtained in the OECD guideline 422 study is mainly based on decrease in food consumption and bodyweight, due to palatability issues with the test item mixed in diet. The only organ-specific adverse effect observed is the well known sex- and species-specific effect in male kidneys linked to alpha-2µglobulin accumulation. As this effect is not relevant to humans, it is not considered for classification of the registered substance. As there is no organ-specific adverse effect observed below the limit of classification (i.e. 300 mg/kg bw/d), the registered substance does not need to be classified for Specific Target Organ Toxicity in Oral Repeated Exposure according to CLP Regulation (1272/2008).

The NOAEC obtained in the OECD guideline 413 study is based on the absence of adverse effects observed at the highest concentration tested. The only organ-specific effect observed is testicular tubular degeneration/atrophy. However, due to the small numbers affected and the absence of findings in the recovery animals, these effects are not considered adverse, it is not considered for classification of the registered substance. As there is no organ-specific adverse effect observed below the limit of classification (i.e. 0.6 mg/L/6h/d), the registered substance does not need to be classified for Specific Target Organ Toxicity in Inhalation Repeated Exposure according to CLP Regulation (1272/2008).

Based on the available information, no additional self-classification is proposed regarding the specific target organ toxicity after oral and inhalation dose-repeated exposures according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

No information is available regarding the dermal route.