Registration Dossier

Administrative data

Description of key information

The key study for repeated dose toxicity is a 90-day subchronic inhalation study with rats and mice for α-pinene which is a major constituent of TOPP (NTP, 2006). The study was conducted in accordance with OECD Test Guideline 413 with deviations (no food consumption, haematology, ophthalmological examination, some organ weights were not recorded).

The No Observed Adverse Effect Concentration (NOAEC) was reported as 200 ppm for females, based on mortality and lower body weight gain at the higher dose level of 400 ppm. No NOAEC was established for males since examination of the male kidneys at all dose levels revealed lesions including granular casts and hyaline droplets indicative of alpha-2u-globulin nephropathy. This is a known male rat-specific effect and is not relevant for human health hazard assessment. The overall NOAEC relevant for humans is therefore 200 ppm (equivalent to 1115 mg/m3).

A number of other studies for the oral and inhalation routes do not indicate that any of the terpene constituents of TOPP for which data are available require classification for specific target organ toxicity following repeated exposures.

Studies with sulfur-containing constituents of TOPP do report adverse effects following repeated inhalation and dermal exposure although none of these is classified for specific target organ toxicity. Furthermore, at the concentrations typically present in TOPP these would not contribute to the overall classification according to the rules for mixtures in Regulation (EC) No 1272/2008.

Conclusions for repeated dose toxicity

The most abundant constituents of TOPP are represented by existing, reliable repeated dose toxicity data and information that is available on minor constituents indicates that serious adverse toxicological effects following repeated exposure are not expected for these types of chemical structures. The available acute toxicity studies for the terpenoid constituents are also consistent with a low hazard profile. The registrants therefore consider that on the grounds of animal welfare and minimising in vivo studies in vertebrates, the conduct of further studies is not warranted for this endpoint. Existing data are adequate to reach appropriate hazard conclusions for repeated dose toxicity.

The most appropriate starting point to calculate the DNEL (workers and general population) for systemic effects via the dermal route is the 90-day inhalatory rat NOAEC for α-pinene (NTP, 2006), 200 ppm or 1115 mg/m3. This constituent is the most abundant in TOPP and for the dermal route, exposure will be to ‘whole’ substance.

The most appropriate starting point to calculate the DNEL (workers and general population) for the inhalation route is the 90-day inhalatory rat NOAEC of ca. 10 ppm (0.039 mg/l) for dimethyldisulfide. Although this substance is only a minor constituent of TOPP in liquid form, its relative proportion in vapour is likely to be higher, this is taken as representative of the whole substance as the worst-case. Refer to Section 5.11 for further discussion on the selection of starting point.

DNELs for the oral and inhalation routes (man via the environment) can be calculated on a block-specific basis. See Section 7.0 Toxicological information for further information.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 June 1996 to 7 October 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
lack of details on doses selection and test substance
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Qualifier:
according to
Guideline:
other: US FDA Toxicological Principles for the Safety Assessment of Direct Food Additives and Colour Additives used in Food
Version / remarks:
(1982)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Hercules Inc., Georgia; FEMA 96-1
- Expiration date of the lot/batch: no data
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored frozen until use
- Stability under test conditions: stable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: thawed in warm water away from light; the test material was weight and added to approx. 200 g feed and mixed for 2 min

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Weight at study initiation: 186 - 255 g for males; 151 - 212 g for females
- Housing: before study initiation two animals of the same sex in a suspended stainess-steel, wire-mesh cage; housed individually after randomization
- Diet: certified rodent diet, ad libitum during the first week of acclimation, then available 12 hours a day.
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY: food was analysed for specified heavy metals and nutrients, aflaxotin, chlorinated hydrocarbons, and organophosphonates, and water was analysed for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 to 24.9°C
- Humidity (%): 40.3 to 68.1 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 13/11
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: thawed in warm water away from light; the test material was weight and added to approx. 200 g feed and mixed for 2 min; the premixes were added to the required amount of feed and mixed in a blender for approx. 1 minute

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): PMI Certified Rodent Diet
- Storage temperature of food: 4°C

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Homogeneity of test material in rodent diet at 0.015 and 3 % was determined in the method validation study
- Stability of test material in rodent diet at 0.015 and 3 % was determined
- Concentration: sample of each formulation prepared on study day 1, 8, 29, 57 and 85 was analysed in duplicate
- Analytical method: used to assay the level of test substance in the rat diet was analysed by extraction of the test article from the diet using acetonitrile solvent followed by high performance liquid chromatography
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
Group 2 animals
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3 animals
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
Group 5 animals: exposed to 300 mg/kg bw/day at week 1, then 600 mg/kg bw/day for the rest of the study period
Dose / conc.:
900 mg/kg bw/day (actual dose received)
Remarks:
Group 6 animals: exposed to 300 mg/kg bw/day during week 1 and 600 mg/kg bw/day during week 2, then to 900 mg/kg bw/day thereafter
No. of animals per sex per dose:
20
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: no data
Positive control:
not used
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations checked in table included: mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to treatment and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: recorded at randomisation, prior to treatment, twice weekly for 4 weeks and weekly there after

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during week 12
- Dose groups that were examined: each animal

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (CO2/O2 anaesthesia)
- Animals fasted: No
- How many animals: each animal
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: No
- How many animals: each animal
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No 2)

HISTOPATHOLOGY: Yes (see table No 2)
Statistics:
Weekly body weights, body weight change, weekly food consumption, total food consumption, efficiency of food utilization, clinical pathology data, terminal body weights, terminal body weights, organ weight data of the treated groups were statistically compared to the control group.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Total body weight change values were statistically lower than the control values for group 3 males, and groups 4 and 5 males and females due to reduced palatability of the diet.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower mean food consumption for groups 3, 4 and 5 males and 4 and 5 females when compared to the control group due to reduced palatability of the diet.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Lower food efficiency for males at 600 and 900 mg/kg bw/day due to reduced palatability of the diet.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Iritis, chromodacryorrhea or retinopathy were observed in four animals, however there was no indication of dose-related ocular disease.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Significant decreases in the mean cell volume and mean cell haemoglobin for group 5 females, and for platelets for group 5 males and females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Higher mean values of gamma glutamyltransferase in group 4 females and group 5 males and females, and alanine aminotransferase and aspartate aminotransferase in group 5 females. Lower mean values of total protein and albumin in group 5 males and females, and glucose in group 4 and 5 females. . The differences were considered incidental to the administration of test compound due to low magnitude of the change and/or lack of dose response.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Increased liver-to-body weight ratio was considered to be related to decreased terminal body weight due to unpalatability of the diet.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Enlarged livers in males and uterine findings in females not considered to be treatment related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hepathocellular hypertrophy characterised by the presence of enlarged hepatocytes with poorly distinguishable cell borders and associated with pale intracytoplasmic material was observed in group 3 males and groups 4 and 5 males and females. The findings were considered to be adaptive and not adverse.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In the 90-day oral toxicity study the reported NOAEL is 300 mg/kg bw/day for male and female rats based on higher levels of gamma glutamyltransferase at 600 mg/kg bw/day and higher doses in females, and increased incidence of single cell necrosis at 600 mg/kg bw/day and higher doses in males.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
16 April 2010 to 03 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
No data
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 104636, supplied by the sponsor
- Expiration date of the lot/batch: 1 March 2010
- Purity test date: 3 March 2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble and stable in corn oil

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No treatment of test material prior to testing
- Final dilution of a dissolved solid, stock liquid or gel: The test substance was prepared as a series of graded concentrations in the vehicle. The required amounts of test material were weighed out and placed in suitable containers. The vehicle was added to the test material and the mixture was magnetically stirred. All formulations were made up using the same method.


Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 303 to 375 g for males; 198 to 253 g for females
- Fasting period before study: The food was withdrawn overnight before routine blood sampling for haematology and blood chemistry.
- Housing: The animals were housed in P2000 or 2154 solid bottomed cages, or RB3 modified cages which were polypropylene cages with stainless steel grid flooring. The animals were housed in RB3 modified cages when paired for mating.
- Diet: standard rodent diet, ad libitum.
- Water: potable water from the public supply, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25°C
- Humidity (%): 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Details on route of administration:
The volume of the received doses was 5 mL/kg.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose levels were selected based on the results of the two week preliminary study which tested dose levels of 150, 600 and 1000 mg/kg bw/day.
VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance was stable in corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initial sampling on each day, the formulation was mixed by 20-fold inversion and magnetic stirring for a minimum of 5 minutes. At each time point, single samples were obtained for assay from the top, middle and the bottom of the magnetically stirred formulation.
Duration of treatment / exposure:
5 weeks
Frequency of treatment:
7 days/ week
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main phase animals: 10 females for each dose; 5 males for control and high dose and 10 males for low and mid doses
Toxicity phase animals: 5 females for all doses
Recovery phase animals: 5 males and 5 females from control and high dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a two-week preliminary study which tested dose levels of 150, 600 and 1000 mg/kg bw/day.
- Rationale for selecting satellite groups: 5 male and 5 female animals from the control and high dose groups were selected for satellite groups.
- Post-exposure recovery period in satellite groups: 2 weeks, vehicle treatment only
Positive control:
Not used
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for evidence of ill-health o0r reaction to the test material

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing commenced and during each week of study for adult animals. For mated females observations were performed on days 0, 7, 14 and 20 after mating and days 1 and 7 of lactation. Detailed physical examinations and arena observations were performed weekly during the recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight were recorded prior to treatment, weekly throughout the dosing and recovery period and before necropsy.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 5 of study, blood samples were obtained before dosing from the first five main phase males and from the toxicity phase females. Blood samples were also obtained from the recovery group at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: five main phase males and five toxicity phase females
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During week 5 of study, blood samples were obtained before dosing from the first five main phase males and from the toxicity phase females. Blood samples were also obtained from the recovery group at the end of the recovery period.
- Animals fasted: Yes
- How many animals: five main phase males and five toxicity phase females
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed on the second five main phase, recovery phase males and on toxicity phase females during week 5 of the study.
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No

OTHER:
OESTRUS CYCLES
- Time schedule for examinations: For 15 days before pairing, daily vaginal smears were taken from all main phase females, using cotton swabs moistened with saline
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No. 2)

HISTOPATHOLOGY: Yes (see table No. 2)
Other examinations:
Mating procedure: During mating period (up to 2 weeks) each morning the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa and the stage of the oestrous cycle.
Parturition observations: from day 20 after mating, main phase females were checked three times daily for evidence of parturition
Statistics:
- ClinAxys II (version 2.0.2) used for haematology and blood chemistry
- Xybion Path/Tox System (version 4.2.2) used for physical examination and arena observations, dosing signs, bodyweights, food consumption, necropsy and pathology data
- Liberate (Release 2) in-house system used for reporting physical examination and arena observations, dosing signs, bodyweights, food consumption, necropsy and pathology data, statistics.
- Agilent Chemstation used for Formulation Analysis.
- Quasar (version 1.1) in-house statistical analysis package used for bodyweight, food consumption and organ weights.
- StarTox (version 3.2) in-house statistical analysis package used for grip strength, motor activity, haematology, blood chemistry, litter size and survival indices.
- SAS (version 9.1.3) statistical analysis package used for sex ratio.
- StatXact 3 (Cytel 1995) statistical analysis package used for gestation length.
- RAMS Rodent Activity Monitoring System (version 2.0) used for activity monitoring
Clinical signs:
no effects observed
Description (incidence and severity):
Some females and males dosed with 750 mg/kg bw/day showed an unsteady gait and underactivity on the first two days of dosing. Overactivity was observed as a post-dosing sign during week 1 in females dosed with 60 mg/kg bw/day and males and females at 250 mg/kg bw/day. Animals dosed with 750 mg/kg bw/day showed signs of salivation and chin rubbing. These are common reactions to the dosing process so were considered unrelated to systemic toxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male animal from 60 mg/kg bw/day group was found dead on day 31. Necropsy revealed that the oesophagus was distended with food material, the trachea contained clear viscous fluid, and the lungs had multiple dark areas and were firm. The cause of the animal's death was considered undetermined.
One female from 60 mg/kg bw/day was killed following difficulties during parturition. Abnormally enlarged placenta was observed at necropsy.
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
During week 5 of dosing, haematocrit, haemoglobin concentrations haematocrit and red blood cell counts were slightly, but significantly lower than Control in females receiving 750 mg/kg/day. Statistical significance was attained for female haemoglobin concentrations in the low and intermediate dose groups but all values were within background range and no dose-related trend was observed. By Week 2 of recovery, all previously affected parameters were essentially similar to those of the controls, indicating complete recovery. Males treated with 750 mg/kg bw/day had statistically significant decrease in haematocrit haemoglobin concentrations and red blood cell count. Howeverthese changes were within the background range and not considered to be of biological significance. Statistically significant longer prothrombin clotting time were noted for recovery males that previously received 750 mg/kg bw/day. These slight changes were not considered to be of biological significance as the animals were not affected during the dosing period and may be associated with the slightly low platelet count for these animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
During Week 5 of study, urea and creatinine levels were significantly higher in females receiving 750 mg/kg/day and slightly, but not significantly, high in males at the same dose level. By Week 2 of recovery urea and creatinine levels were still higher than control for males and females previously receiving 750 mg/kg/day, attaining statistical significance for females only. Glucose plasma levels were significantly higher than control in females dosed at 250 and 750 mg/kg/day. Potassium levels were significantly higher than control in males and females receiving 750 mg/kg/day. Glucose plasma levels and potassium levels were similar to respective controls during the recovery period. Bile acid plasma levels for females at all dose levels were higher than the concurrent control attaining significance at 750 mg/kg/day. A dose-related trend was apparent, however, individual values were all within the historical control range.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Testis weight was markedly lower in males receiving 750 mg/kg/day (58% of Control) and there was also a suggestion of slightly lower epididymal weights for these males. After two weeks without dosing testis and epididymal weights showed no evidence of recovery.
Mean liver weights of males and females dosed with 750 mg/kg bw/day were significantly increased. Kidney weights of males dosed with 750 mg/kg bw/day were also significantly increased. Liver and kidney weights were no longer enlarged after a 2-week recovery period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 750 mg/kg/day showed a range of testicular findings including small, blue and flaccid testes. Epididymides of males receiving 750 mg/kg/day were observed generally small but some contained masses or appeared enlarged. These findings were still evident after two weeks of recovery.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Centrilobular hepatocyte hypertrophy was observed in females dosed with 750 mg/kg bw/day. These effects were not observed after a 2-week recovery period. Cortical tubules with hyaline droplets and cortical tubular basophila in kidneys were observed in males dosed with 750 and 250 mg/kg bw/day. Granular casts were seen in two males treated with 750 mg/kg bw/day. Complete recovery was seen after a 2-week recovery period. Absence of spermatozoa was observed in 4 males dosed with 750 mg/kg bw/day. Degenerate spermatogenic cells in ducts were noted in all males treated with 750 mg/kg bw/day. Moderate to severe seminiferous tubular atrophy and minimal to slight seminiferous tubular vacuolation were seen in all animals dosed with 750 mg/kg bw/day. Minimal to moderate spermatid giant cells were also seen in all males treated with 750 mg/kg bw/day. Test article related changes were present in the epididymides and testes of males administered 750 mg/kg bw/day of test material after the recovery period. No test material-related changes were observed in the tissues from females given 750 mg/kg bw/day killed on day 25 of the gestation phase due to suspect fertility.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Food consumption: increased food consumption in all animals during the recovery period was due to cessation of dose vadministration which used corn oil as the vehicle thereby supplying a portion of the required nutrients.
Water consumption: animals dosed with 750 mg/kg bw/day consumed more water than the controls.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
Conclusions:
In the combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test the reported NOAEL for was 250 mg/kg bw/day based on testicular and epididymal toxicity in male rats treated with 750 mg/kg bw/day.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 November 2011 to 27 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 119444
- Expiration date of the lot/batch: 15 June 2012
- Purity test date: 16 June 2011
- Impurities (identity and concentrations): gamma terpinene (0.7%), D and L limonene (0.1%)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4ºC in the dark, under nitrogen
- Stability under test conditions: the dietary admixtures were stable for a period of three weeks at approximately -18°C
- Solubility and stability of the test substance in the solvent/vehicle: soluble in corn oil

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was initially mixed with 2 % corn oil and subsequently a small amount of basal laboratory diet was incorporated until homogenous. The premix was then added to a larger amount of basal laboratory diet and mixed for 19 minutes at a constant speed.
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD® BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River ltd., UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 358 to 409 g for males, 210 to 264 g for females
- Housing: Initially the animals were housed in groups of five in solid floor polypropylene cages with stainless steel mash lids and softwood flake bedding. During the mating phase the animals one main phase female was housed with one main/recovery phase male in polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: a ground diet, ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 5 %
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 January 2012 (first day of treatment). To: 02 March 2012 (final necropsy)

Route of administration:
oral: feed
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was initially mixed with 2 % corn oil and subsequently a small amount of basal laboratory diet was incorporated until homogenous. The premix was then added to a larger amount of basal laboratory diet and mixed for 19 minutes at a constant speed.

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 (Type of food): Rodent PMI 5002 (Certified)
- Storage temperature of food: The diet was stored in labelled, double plastic bags at approximately -18°C

VEHICLE
- Justification for use and choice of vehicle (if other than water): to avoid evaporation
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data
- Purity: no data
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. Chromatography was used for analysis. Results indicated that the mean prepared dietary admixture concentrations were within acceptable ranges for the purpose of this study.
Duration of treatment / exposure:
up to 56 days:
Main phase: males were dosed daily during premating and mating periods and up to 42 days; females were dosed up to 56 consecutive days (including a three week maturation phase, pairing, gestation and early lactation for females).
Toxicity phase: females were dosed daily up to 42 consecutive days.
Recovery phase: recovery phase animals were treated with the high dose or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days.
Frequency of treatment:
one daily, 7 days a week
Dose / conc.:
294.6 mg/kg bw/day (actual dose received)
Remarks:
Mean achieved dose in males at 5000 ppm in diet
Dose / conc.:
312.5 mg/kg bw/day (actual dose received)
Remarks:
Mean achieved dose in females at 5000 ppm in diet
Dose / conc.:
142.9 mg/kg bw/day (actual dose received)
Remarks:
Mean achieved dose in males at 2500 ppm in diet
Dose / conc.:
161.5 mg/kg bw/day (actual dose received)
Remarks:
Mean achieved dose in females at 2500 ppm in diet
Dose / conc.:
49.5 mg/kg bw/day (actual dose received)
Remarks:
Mean achieved dose in males at 800 ppm in diet
Dose / conc.:
56.5 mg/kg bw/day (actual dose received)
Remarks:
Mean achieved dose in females at 800 ppm in diet
No. of animals per sex per dose:
main phase males: 5 rats in the high dose and ten rats in the low and intermediate group
main phase females: 10 rats for each dose
toxicity group females: 5 rats for each dose
recovery groups: 5 males and 5 females
Control animals:
other: yes, concurent diet with 2 % corn oil
Details on study design:
- Dose selection rationale: dose levels were chosen based on the results of previous toxicity study
- 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: animals treated with the highest dose and control groups, to assess reversibility of effects
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations included: signs of toxicity, ill-health and behavioural change

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to treatment and once weekly thereafter for main phase males, toxicity phase females and recovery animals. Weekly observations were performed on main phase females during the pre-mating period, then on days 0, 6, 13, 20 post coitum and on days 1 and 7 of lactation.

BODY WEIGHT: Yes
- Time schedule for examinations: on day 1 and then weekly for main phase males and toxicity phase females until termination; on day 1 and then weekly until pairing for main phase females; on days 0, 6, 13 and 20 post coitum and on days 1, 4 and 7 post partum for mated females; on day 1 and then weekly for recovery animals

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination on day 42 for maihn phase males and toxicity phase females; on termination day 56 for recovery group animals
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: 5 males from main phase animals and 5 females from toxicity group and all recovery group animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination on day 42 for maihn phase males and toxicity phase females; on termination day 56 for recovery group animals
- Animals fasted: No
- How many animals: 5 males from main phase animals and 5 females from toxicity group and all recovery group animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the final week of treatment
- Dose groups that were examined: the first five main phase males per dose group and in toxicity phase females
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No 2 )

HISTOPATHOLOGY: Yes (see table No 2)
Statistics:
The following parameters were subjected to statistical analysis: quantitative functional performance data; body weight and body weight change; food consumption during gestation and lactation; pre-coital interval and gestation length; litter size and litter weights; sex ratio; implantation sites; implantation loss and viability indices; offspring body weight and body weight change; haematology, blood chemistry, adult absolute and body weight-relative organ weights
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs that were considered to be treatment-related were observed. One toxicity phase female treated with 5000 ppm had hunched posture on Day 41 only. 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.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced overall body weight gain was evident in animals of either sex treated with 5000 ppm and in females treated with 2500 ppm. Statistically significant reductions in gestation and lactation body weights were evident in 5000 ppm main phase females and consequently statistically significant reductions in body weight gain during lactation were evident in these females. Both main phase males and females showed statistically significant reduction in body weight gain during the first week of treatment at 2500 ppm and 800 ppm, respectively.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Reduced dietary intake in all the animals treated with 5000 ppm during the first week of treatment. Toxicity and recovery phase females showed a slight reduction in food consumption throughout the remaining treatment period. Main phase females treated with 5000 ppm showed a statistically significant reduction in dietary intake throughtout gestation and lactation. A statistically significant reduction in dietary intake was also evident in main phase females treated with 2500 ppm during the first two weeks of gestation. No such effects were detected in males treated with 2500 or 800 ppm. Females treated with 800 ppm showed a statistically significant increase in food consumption during gestation. These changes were not considered to be treatment-related.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Food efficiency was reduced in animals of either sex treated with 5000 ppm. Reductions were evident during Weeks 1, 2, 4 and 5 for males and during Weeks 1 to 3 for females. No such effects were detected in animals of either sex treated with 2500 or 800 ppm.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Main phase males treated with 5000 ppm showed a statistically significant reduction in activated partial thromboplastin time. Toxicity phase females from this treatment group showed a statistically significant increase in erythrocyte count. Recovery 5000 ppm females showed a statistically significant increase in haemoglobin and haematocrit following fourteen days without treatment. Recovery 5000 ppm males showed a statistically significant increase in eosinophil count. These intergroup differences were considered to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Main phase males treated with 5000 and 2500 ppm showed a statistically significant increase in urea. Main phase males treated with 5000 ppm showed a statistically significant increase in total protein whilst toxicity phase females from this treatment group showed a statistically significant increase in bilirubin. The majority of individual values were within the normal ranges for rats of the strain and age used, therefore the intergroup differences were considered to be of no toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects in behavioural assessments, functional performance or sensory reactivity. Toxicity phase females from all treatment groups showed a statistically significant
increase in mean hind limb grip strength. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the finding was considered to be of no toxicological significance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Main phase males treated with 5000 ppm showed statistically significant increase in absolute and relative liver weight. These changes were also evident in the 5000 ppm recovery group males. No toxicologically significant effects were detected in any treated female or in males treated with 2500 or 800 ppm. Statistically significant reductions in epididymides and putuitary absolute and relative weights were noted for main phase males from all treatment groups. Reduction in absolute and relative heart weight was also noted for males treated with 800 ppm. Toxicity phase females treated with 5000 and 2500 ppm showed statistically significant reduction in absolute and relative thyroid weight. The majority of individual values for epididymides, heart, and thyroid weights were within the normal ranges for rats of this strain and there were no findings at histopathology. Therefore, these findings wee considered to be of no toxicological significance. Main phase males treated with 5000 and 2500 ppm also showed a statistically significant reduction in relative and absolute seminal vesicle, thyroid and prostate weight. Statistically significant reduction in relative and absolute adrenal and pituitary weights was noted in toxicity group females treated with 5000 and 2500 ppm. The majority of individual values for adrenal and thyroid weights were within the normal range for animals of this strain and considering the absence of changes at histopathology these findings were of no toxicological significance. Statistically significant reduction in absolute brain and liver weight was observed for recovery 5000 ppm females and in absolute thyroid weight for recovery 5000 ppm males. In the absence of similar effects detected in main phase males or toxicity phase females at the end of the treatment period, the intergroup differences were considered to be of no toxicological significance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
One toxicity phase female treated with 5000 ppm had a mottled liver at necropsy. There were no findings in the liver during histology, thus changes in liver were considered not to be of toxicological significance. One recovery 5000 ppm male had an enlarged mandibular lymph node at necropsy whilst one recovery 5000 ppm female had reddened lungs at necropsy. No such effects were observed in animals at the end of the treatment period and these effects were considered to be of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal to slight centrilobular hepatocellular hypertrophy was evident in all main phase males treated with 5000 and 2500 ppm. The hepatocellular hypertrophy was partly reversible in severity but was still noted in all five recovery males at 5000 ppm at minimal severity. Minimal to marked multifocal tubular degeneration/regeneration in the renal cortical tubules was evident in four out of five main phase males at 800 ppm and in all main phase males at 2500 and 5000 ppm. In all these males slight to marked hyaline droplets were present in the proximal convoluted tubules and in one male at 800 ppm, two males at 2500 ppm and in four males at 5000 ppm minimal to moderate granular casts occurred in the tubules of the inner cortex. The focal to multifocal tubular basophilia present incidentally in some males at 0, 800 and 2500 ppm was not evident at 5000 ppm. After recovery the tubular basophilia was evident at minimal severity in three of the controls and in all recovery males at 5000 ppm. Minimal to slight multifocal tubular degeneration in renal cortical tubules was evident in four of the recovery males at 5000 ppm. In all of these males minimal to slight hyaline droplets were present in the proximal convoluted tubules. Granular casts were also present in four of the recovery males at 5000 ppm.
Histopathological findings: neoplastic:
no effects observed
Details on results:
In terms of extrapolation to man and risk assessment calculations whereby effects relating to male rat renal changes are species and sex specific and therefore are not relevant.
Key result
Dose descriptor:
NOAEL
Effect level:
294.6 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed at 5000 ppm for male and female rats.
Key result
Dose descriptor:
NOAEL
Effect level:
312.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed at 5000 ppm for male and female rats
Key result
Critical effects observed:
no
Conclusions:
In the combined repeat dose toxicity study with reproduction/ development toxicity screening test in the rat the reported NOAEL value for male and female rats is 2500 ppm (154.6 mg/kg bw/day). However, the study reviewer concluded a NOAEL of 5000 ppm for systemic toxicity since the observed effect was reduced food consumption in high dose males and females which caused reduction in body weigh gain.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 weeks in 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
food consumption, hematology, ophthalmological examination, some organ weights were not recorded.
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
food consumption, hematology, ophthalmological examination, some organ weights were not recorded
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups.
Route of administration:
inhalation
Type of inhalation exposure:
not specified
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
14 weeks; 6 hours per day
Frequency of treatment:
five times per week, weekdays only
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
No data
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes for moribundity and death
- Time schedule: twice daily, at least 6 hours apart (before 10:00 AM and after 2:00 PM)

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 of the test, after 7 days and at weekly intervals thereafter

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
no data
Statistics:
Kaplan-Meier used for probability of survival. Statistical analyses used for possible dose-related effect on survival was Cox (Cox D.R. (1972) Regression models and life tables. J.R. Stat. Soc. B34: 187-220.) for testing two groups for equality; and Tarone’s (Tarone R.E. (1975) Tests for trend in life table analysis. Biometrika 62; 679-682) life table test for a dose-related trend.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
MORTALITY
In the high dose group (400 ppm), 6 females were found dead before the end of the study (4 on Day 36, 1 on Day 50 and 1 on Day 91). All other animals survived until terminal sacrifice.

CLINICAL EXAMINATION
Three females of the high dose group (which survived until terminal sacrifice) displayed mild tremors.

BODY WEIGHT AND WEIGHT GAIN
Males: during the first week of treatment, the body weight gain was slightly lower in all treated groups when compared to the control group. The overall bodyweight gain was significantly lower in the high dose group (197 g vs 224 g in the control group).
Females: during the first week of treatment, the body weight gain was lower in the high dose group only when compared to the treated group. Thereafter the mean bwg of females in the high dose group was variable but overall approximately half this of the control group. All surviving females at 400 ppm lost weight between week 12 and week 14.

CLINICAL CHEMISTRY
Males showed statistically significant reductions in sorbitol dehydrogenase activity at 400 ppm, alanine aminotransferase activity at levels ≥50 ppm, and alkaline phosphatase activity at levels ≥100 ppm. Females showed statistically significant reductions in alanine aminotransferase activity at levels ≥200 ppm, and alkaline phosphatase activity at the 400 ppm. There were significant decreases at lower levels of exposure for females but these changes were not dose-dependent. None of these changes in enzyme activity were related to organ weight changes or evidence of histopathology.

ORGAN WEIGHTS
Absolute and relative liver weights were statistically increased in males at 200 ppm and greater and relative and absolute kidney weights were increased in males at 100 ppm and greater. In females, relative and absolute liver weights were increased at levels of ≥50 ppm, but there were no increases in either hepatic enzymes or any evidence of histopathological changes at any of these dose levels.
Females showed statistically significant decreases in absolute and relative thymus weights and increased relative lung weight at the 400 ppm level.

HISTOPATHOLOGY: NON-NEOPLASTIC
Examination of the male kidneys at all dose levels revealed lesions including granular casts and hyaline droplets indicative of α2u-globulin nephropathy.
In females there was no evidence of histopathology in any organ at any dose level. Specifically, there was no evidence of histopathological changes to the clitoris, ovaries, uterus, epididymis, preputial gland, seminal vesicles, and testes for any of the control or test groups of animals.

Key result
Dose descriptor:
LOAEL
Effect level:
25 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Nephropathy which is relevant only in male rats. Lower body weight gain in the high dose group. In humans, this LOAEL will not be relevant.
Key result
Dose descriptor:
NOAEL
Effect level:
200 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on mortality and lower body weight gain in the high dose group.
Critical effects observed:
no

Table 1 - Mean body weight gain (g) in males

Dose Group Wk 1-2 Wk 1-14
Vehicle Control 55.6 224
25 ppm 45.7 212
50 ppm 49.2 221
100 ppm 36.6 207
200 ppm 46.0 216
400 ppm 33.2 197

Table 2 - Mean body weight gain (g) in females

Dose Group Wk 1-2 Wk 1-14
Vehicle Control 26.4 99
25 ppm 25.1 101
50 ppm 27.5 109
100 ppm 19.0 99
200 ppm 22.7 105
400 ppm 16.2 57
Conclusions:
The LOAEL in male rats is the lowest dose level tested, but it is not relevant to humans as it is based on renal effects linked to alpha2u-globulin accumulation. When considering effects other than those on kidneys in males, a lower body weight gain was observed at 400 ppm when compared to controls.
A NOAEL could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain at the next dose level when compared to controls.

The overall NOAEL relevant for humans is 200 ppm.
Executive summary:

In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 weekdays per week at 0, 25, 50, 100, 200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was performed on all animals, including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.

Apart from lesions including granular casts and hyaline droplets indicative of alpha2u-globulin nephropathy observed in all treated group males, a lower body weight gain than in controls was observed in the high dose group in both sexes. In addition, 6 females died and 3 females displayed mild tremors in the high dose group too.

The LOAEL in male rats is the lowest dose level tested, but it is not relevant to humans as it is based on renal effects linked to alpha2µ-globulin accumulation. When considering effects other than those on kidneys in males, a lower body weight gain was observed at 400 ppm when compared to controls. A NOAEL could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain at the next dose level when compared to controls. The overall NOAEL relevant for humans is 200 ppm.

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 weeks in 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
food consumption, hematology, ophthalmological examination, some organ weights were not recorded.
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
food consumption, hematology, ophthalmological examination, some organ weights were not recorded
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups.
Route of administration:
inhalation
Type of inhalation exposure:
not specified
Vehicle:
not specified
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
14 weeks; 6 hours per day
Frequency of treatment:
Five times per week, weekdays only
Dose / conc.:
25 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
Dose / conc.:
200 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
No
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes for moribundity and death
- Time schedule: twice daily, at least 6 hours apart (before 10:00 AM and after 2:00 PM)

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 of the test, after 7 days and at weekly intervals thereafter

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
No data
Statistics:
Kaplan-Meier used for probability of survival. Statistical analyses used for possible dose-related effect on survival was Cox (Cox D.R. (1972) Regression models and life tables. J.R. Stat. Soc. B34: 187-220.) for testing two groups for equality; and Tarone’s (Tarone R.E. (1975) Tests for trend in life table analysis. Biometrika 62; 679-682) life table test for a dose-related trend.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
MORTALITY
All mice survived until the terminal sacrifice.

CLINICAL EXAMINATION
There were no treatment-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was comparable for all test animals when compared to controls.

CLINICAL CHEMISTRY
No data available.

ORGAN WEIGHTS
Absolute liver weights were increased for both sexes at the 400 ppm and relative and absolute liver weights were increased for both sexes at 200 ppm and 400 ppm. The 400 ppm male group showed decreased absolute and relative thymus weight. No gross or microscopic lesions were associated with these organ weight findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination of male and female mice exposed to atmospheres of ≥100 ppm of α-pinene revealed evidence of hyperplasia of the transitional epithelium of the urinary bladder. However, there was no evidence of histopathological changes to the clitoris, ovaries, uterus, epididymis, preputial gland, seminal vesicles, and testes any of the control or test groups of animals.

Dose descriptor:
NOAEL
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Presence of transitional cell hyperplasia of the urinary bladder.
Critical effects observed:
not specified

Table 1 - Histopathological effects - Urinary bladder - Males

Dose Group Urinary bladder - Transitional epithelium - Hyperplasia
Number of animals
  Absent Minimal Mild Moderate
Vehicle Control 10 0 0 0
25 ppm 10 0 0 0
50 ppm 10 0 0 0
100 ppm 3 6 1 0
200 ppm 0 0 10 0
400 ppm 0 0 5 5

Table 2 - Histopathological effects - Urinary bladder - Females

Dose Group Urinary bladder - Transitional epithelium - Hyperplasia
Number of animals
  Absent Minimal Mild Moderate
Vehicle Control 10 0 0 0
25 ppm 10 0 0 0
50 ppm 10 0 0 0
100 ppm 4 6 0 0
200 ppm 0 4 6 0
400 ppm 0 0 8 2
Conclusions:
The NOAEL in male and female rats is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated at 100 to 400 ppm.
Executive summary:

In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 weekdays per week at 0, 25, 50, 100, 200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was performed on all animals including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.

Similar effects were observed in male and female mice from the same dose level (100 ppm): minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder.

The NOAEL in male and female rats is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated at 100 to 400 ppm.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
38.5 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
38.5 mg/m³
Study duration:
subchronic
Species:
rat

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

No reliable repeated dose toxicity studies are available for TOPP. A reliable subchronic vapour inhalation study is, however, available for the major constituent, α-pinene, which comprises between 10 and 85% by weight of TOPP.

Lower concentrations (up to around 5% each) of other terpenes are present. These terpene constituents are also present in varying proportions in other forms of turpentine, all of which are captured in the general description of CAS Number 8006-64-2. Reliable subacute and subchronic oral toxicity data are available for a number of these, as presented in Table 5.6.1 and discussed further below.

Relevant data for a number of other constituents, including the sulfur-containing constituents, and these are also taken into consideration in order to reach an appropriate overall hazard conclusion for repeated dose toxicity of TOPP.

As described in Section 1.3 of the Chemical Safety Report, constituents of TOPP can be grouped into ‘blocks’ according to chemical structure and properties and these are shown in Table 5.7.3 below along with a summary of available repeated dose toxicity data for each block.

Table 5.7.3 Available repeated dose toxicity data for TOPP Blocks

Block number

Block name

Typical %wt in TOPP

Representative constituents with repeated dose toxicity data

1

Pinene Block

71.5

α- Pinene (inhalation route)

2

Delta-3-carene Block

13

No data available; REACH registration uses read-across from α- Pinene

3

Dipentene Block

7.8

Terpinolene (oral route)

4

Dimethyl sulfide Block

1.2

Dimethyl sulfide (inhalation and oral routes)

5

Methyl mercaptan Block

0.06

Methyl mercaptan (inhalation route)

6

Sesquiterpes Block

4.3

No data available

7

Terpene alcohols Block

1.0

Terpineol multi (oral route)
Anethole (oral route)

8

Camphene Block

0.81

Camphene (oral route)

9

Dimethyl disulfide Block

0.32

Dimethyl disulfide (inhalation route)

 

Inhalation route

Alpha-pinene (CAS 80-56-8)

A sub-chronic inhalation toxicity study in rats and mice (NTP, 2006) is available for the major constituent of TOPP, alpha-pinene (CAS 80-56-8). This constituent is present at 10 – 85% by weight in the registration substance (typical concentration 57%). The study was largely in accordance with OECD Test Guideline 413, but with some deviations relating to food consumption, haematology, opthalmology and organ weight examinations. The study was therefore assigned Klimisch score 2.

In the test with rats, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 weekdays per week at 0, 25, 50, 100, 200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was performed on all animals, including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.

Apart from lesions including granular casts and hyaline droplets indicative of alpha-2u-globulin nephropathy observed in all treated group males, a lower body weight gain than in controls was observed in the high dose group in both sexes. In addition, 6 females died and 3 females displayed mild tremors in the high dose group too.

The LOAEC in male rats is the lowest dose level tested, but it is not relevant to humans as it is based on renal effects linked to alpha-2u-globulin accumulation. When considering effects other than those on kidneys in males, a lower body weight gain was observed at 400 ppm when compared to controls. A NOAEL could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain at the next dose level when compared to controls. The overall NOAEC relevant for humans is 200 ppm.

In the test with mice, groups of 10 animals per dose and per sex were administered for 6 hours per day, 5 weekdays per week at 0, 25, 50, 100, 200 and 400 ppm for a total of 14 weeks. The animals were observed twice per day and weighed once per week. A complete histopathologic evaluation including treatment-related gross lesions was performed on all animals including early death animals. Treatment-related lesions (target organs) were identified and these organs and gross lesions were examined to a no-effect level.

Similar effects were observed in male and female mice from the same dose level (100 ppm): minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder.

The NOAEC in male and female rats is 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder in animals treated at 100 to 400 ppm.

The test with rats is assigned as the key study, since the EU classification criteria for repeated dose toxicity are based on this test species.

Dimethyl disulfide (CAS 624-92-0)

The REACH dissemination dossier for dimethyl disulfide reports a number of key repeated inhalation studies. In a 90-day repeated dose inhalation study on rats (OECD Test Guideline 413), rats were exposed whole body to 0, 10, 50, 150, or 250 ppm (approximately 0, 0.039, 0.19, 0.578 or 0.963 mg/l day, respectively) DMDS for 6 hours per day for 90 days. Satellite groups were evaluated following a 2-week recovery period. The overall NOAEC was determined to be ca. 10 ppm (0.039 mg/l) based on reversible microscopic changes in nasal mucosa (local effect). The LOAEC was 50 ppm (0.19 mg/l) based on minor effects in blood chemistry and changes in nasal mucosa.

In a study which was carried out following the OECD Test Guideline 413, groups of 10 F344 rats of each sex were exposed to dimethyl disulfide (DMDS) vapor by whole-body exposure at concentrations of 0, 5, 25, or 125 ppm for 6 h/day, 5 days/wk for 13 wk. At 25 ppm, a decrease in the body weight gain and food intake was observed in the males, but not in the females. However, at 125 ppm, a decrease in the body weight gain, food intake, and thymus weight and an increase in the weights of adrenal glands were observed in both genders. In contrast, no treatment-related effects were observed in the 5 ppm group. In these experimental conditions, the target organ was not determined in rats. The no-observed-adverse-effect concentration (NOAEC) was found to be 5 ppm, 6 h/day for male rats and 25 ppm, 6 h/day for female rats.

In addition, a rat neurotoxicity study performed in accordance with OECD Test Guideline 424 is reported. Following exposure to DMDS vapour for 6 hours per day for 13 weeks at concentrations of 0, 5, 20 and 80 ppm, the no-observed-adverse-effect concentration (NOAEC) for systemic toxicity and neurotoxicity was 20 ppm for males and 80 ppm for females. The NOAEC for local contact (nasal) irritation was 5 ppm for both males and females.

The observed effects were not sufficient to trigger classification of dimethyl disulfide for specific target organ toxicity following repeated exposure. However, a proposal for harmonised classification and labelling includes classification for respiratory tract irritation (STOT SE 3; H335: May cause respiratory irritation).

Dimethyl sulfide (CAS 75-18-3)

Limited details are available of a 6-month inhalation study (Food and Cosmetics Toxicol. 17 (4), 1979) cited in IUCLID 2000) in which the LOAEC was apparently 25 mg/m3 and the NOAEC 5 mg/m3.

Methanethiol (methylmercaptan, CAS 74-93-1)

A 90-day inhalation study in rats on methyl mercaptan (CAS 74-93-1) gave a No Observed Adverse Effect Concentration (NOAEC of 17 ppm, equivalent to 0.034 mg/l (Tansy et al., 1981, cited US Public Health Service, 1992) based on decreased body weights at the higher dose of 57 ppm. The same study is referenced in IUCLID 2000, quoting a Lowest Observed Adverse Effect Concentration (LOAEC of 2 ppm, based on pathological changes in the liver. The authors of the study stated that results of blood chemistry studies (i.e., increased total protein with decreased serum albumin) were suggestive of liver damage, but that dehydration could not be ruled out as the cause. Based on the limited information available, the observed effects would not be considered significant for classification purposes.

Oral route

Terpinolene/Iso terpinolene (CAS 586-62-9)

Terpinolene and isoterpinolene (both assigned CAS number 586-62-9) are each present in TOPP at concentrations in the range 0 – 5% by weight. In the blocking approach used for hazard and exposure assessment (CSR Section 1.3), these constituents are members of Block 3 (Dipentene block) which in total typically represents approximately 7% by weight of the registration substance. In a combined repeated dose toxicity study with a reproduction / developmental toxicity screening test (Harlan Laboratories, 2014a) conducted according to OECD Test Guideline No 422 and in compliance with GLP, three groups of Sprague-Dawley Crl: CD®BR strain rats, each comprising of ten males and ten females for the main phase (except for control and top dose: 5 males/dose), five females for the toxicity phase and 5 males and 5 females/dose (control and top dose) for the recovery phase received high purity terpinolene monoconstituent at dietary concentrations of 0, 800, 2500 or 5000 ppm (initially mixed with 2% corn oil). Main phase males and toxicity phase females were dosed daily for 42 days. Recovery phase animals were treated with the high dose or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption, haematology, blood chemistry, oestrous cycle, organ weight, macroscopic and microscopic pathologyinvestigations. There were no unscheduled deaths during the study, and no clinical signs attributed to the test item were recorded. The results of behavioural, functional performance and sensory reactivity assessments were normal. Body weight gain and food consumption were decreased in males and females treated with 5000 ppm receiving 5000 ppm (-24% males, -50% females). The reduced dietary intake was considered by the authors of the report to be due to low palatability of the diet admixture, therefore the effects on body weight gain and food consumption are not considered by the reviewer to be adverse. Minimal effects detected in females treated with 2500 ppm (161.5 mg/kg bw/day) (isolated reduction in by weight gain and food consumption) were considered not to represent an adverse effect of treatment. Main phase males treated with 5000 ppm showed statistically significant increase in absolute and relative liver weight. These changes were also evident at the end of the recovery period in the 5000 ppm recovery group males. Minimal to slight centrilobular hepatocellular hypertrophy was evident in main phase males treated with 5000 and 2500 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. The changes in the liver are considered by the reviewer to be adaptive, and therefore not adverse. Kidney effects including hyaline droplets, granular casts and minimal to marked multifocal tubular degeneration/regeneration in the renal cortical tubules was evident in main phase males from all treatment groups. These kidney effects are compatible with alpha-2μ-globulin nephropathy and are not found in immature rats, female rats or humans and therefore are considered to be of no toxicological importance or relevance to man. It was therefore concluded that the NOAEL for male and female rats was considered to be 5000 ppm, 289 mg/kg bw/day (male), 298mg/kg bw/day (female).

Terpineol Multi (CAS 8000-41-7)

In the blocking approach used for hazard and exposure assessment (CSR Section 1.3), terpene alcohol constituents are members of Block 7 which in total typically represents approximately 1% by weight of the registration substance. The test substance Terpineol Multi (CAS 8000-41-4) is a multiconstituent substance containing predominantly α-terpineol (CAS 98-55-5; 0 – 5% by weight of TOPP) with smaller amounts of β-terpineols, γ-terpineol, δ-terpineol, terpinen-1-ol-4, terpinen-3-ol-1, borneol, isoborneol, fenchol and other minor constituents. Please refer to the confidential test material information in the IUCLID dataset for further details on the composition of the test material. A study is available in which Terpineol Multi was administered daily for at least five weeks, according to OECD 422 and in compliance with GLP (Huntingdon Life Sciences, 2010). Crl:CD(SD) rats in three groups, each comprising ten male and female rats (reproductive phase) and five female rats (toxicity phase) were dose by oral gavage with 60, 250 or 750 mg/kg bw/day. An additional satellite group of five males and five females were dosed daily with the vehicle or at 750 mg/kg bw/day for five weeks and then given two weeks of recovery before termination. Two unscheduled deaths occurred, one male and one female, which were not attributed to test substance. There were no obvious treatment-related findings from clinical observations, detailed physical examination observation or behavioural testing. Relative and absolute liver weights were increased in top dose males and females, accompanied by centrilobular hepatocyte hypertrophy in females. Relative kidney weights were increased in top dose males, and histopathological changes in the kidneys of males at 250 and 750 mg/kg bw/day were recorded. Liver and kidney changes resolved after 14 days in the satellite group animals receiving test substance. There were no significant haematological findings. Urea and creatine levels were significantly higher in top dose females, and were still elevated at the end of the recovery period. Glucose levels in mid- and high-dose females and potassium levels in high-dose males and females were also elevated, but were similar to controls in the recovery phase. The most significant finding was changes in relative and absolute testis and epididymal weights in high dose males. Gross necroscopy showed a range of testicular findings including small, blue and flaccid testes. Microscopic examination revealed reduced numbers or complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) were observed in the epididymides of males receiving 750 mg/kg/day. The effects observed in male reproductive organs persisted until the end of the recovery period. It was concluded that the NOAEL for systemic toxicity for unmated females and for reproductive toxicity for males was 250 mgkg bw/day.

Trans-Anethole (CAS 4180-23-8)

Trans-anethole (CAS 4180-23-8) may be present in TOPP at up to 1% by weight and is another representative of Block 7 (terpene alcohols). A study (OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents).

Based on the findings which are directly related to treatment, excluding those resulting from the diminishing palatability of the diet at higher doses, the NOAEL is 300 mg/kg/day for females and male rates (based on an elevation in serum gamma glutamyltransferase) at ≥600 mg/kg/day in females, and an increased incidence of single cell necrosis at ≥600 mg/kg/day in males) (Corning Hazleton, 1997).

In a chronic repeated dose oral toxicity study in rats, similar to OECD Test Guideline 452 (Chronic Toxicity Studies), the NOAEL of trans-anethole was determined to be greater than the highest dose tested (2500 ppm) as no effects were reported during the conditions of the study (Hagan et al., 1967).

In a study in rats similar to the OECD Test Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies) no apparent reactions to treatment with trans-anethole were noted. Only the animals that died or were killed when moribund showed clinical signs, such as decreased body weight, anorexia and lethargy. No neoplastic lesions were reported during the conditions of the study (Truhaut et al., 1989).

Camphene (CAS 79-92-5)

Camphene is present in TOPP at 0 – 5% by weight and represents Block 8 (camphene). A 28-day repeated dose oral toxicity study, conducted according to OECD 407 and in compliance with GLP, is reported in the disseminated REACH dossier [2] for this substance. Groups of 5 male and 5 female Wistar rats were dosed by oral gavage with 62.5, 250 and 1000 mg/kg bw/day. In the highest dose group, relative and absolute liver weights were increased, and increased vacuolization of the hepatocytes was observed. These effects might be a result of adaptive changes, but as it was not possible to obtain access to the study report, this could not be determined. In male rats, effects were seen in the kidneys at all doses, which are consistent with alpha-2μ-globulin nephropathy, an effect which is only observed in immature male rats and is of no toxicological importance or relevance to man. The disseminated dossier reports a conclusion of NOEL of 250 mg/kg bw for females and LOEL of 62.5 mg/kg bw for males (the lowest dose tested), however as the effects in males are not relevant to humans the NOAEL appropriate to be used in risk assessment is 250 mg/kg bw/day.

Dimethyl sulfide (CAS 75-18-3)

A 14-week oral toxicity study on dimethyl sulfide (75-18-3) gave a NOAEL of 250 mg/kg bw/day (Butterworth et al., 1975, cited in IUCLID 2000).


[1] Accessed 7th November 2016

[2] Accessed 8th November 2016

Justification for classification or non-classification

None of the repeated dose toxicity studies with constituents of TOPP showed effects that were of significance for classification for specific target organ toxicity following repeated exposures.