Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Lime Oxide
- Molecular formula: predominantly a mixture of monoterpenes C10H16 and cyclic monoterpene ethers C10H18O
- Molecular weight: monoterpenes C10H16 molecular weight 136.2, cyclic monoterpene ethers C10H18O molecular weight 154.2
- Physical state: clear colorless liquid
- Analytical purity: 61.8%, sum of 4 major peaks
- Impurities: Known impurities are similar in nature to the major components and have empirical formulae of either C10H16 or C10H18O
- Lot/batch No.: SC00009577
- Expiration date of the lot/batch: 13 October 2015
- Storage condition of test material: room temperature, in the dark
Specific details on test material used for the study:
The test article is an aroma chemical that requires additional information for REACH registration.
The high dose was selected to indicate the presence/absence of toxicity at the limit dose.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
A sufficient number of male and female Crl:WI(Han) strain rats were obtained from Charles River Laboratories, Margate, UK, in order to provide the correct number of animals for study selection.
Sex:
male/female
Details on test animals and environmental conditions:
The animals were housed in cages that conform to the 'Code of practice for the housing and care of animals used in scientific procedures' (Home Office, London, 1989).
Animals were housed in groups of two (males and pre-pairing females), one female with one male (pairing) or singly (mated females).
Relevant animals were housed individually for approximately 24 hours prior to Functional Observational Battery (FOB) assessment.
During the mating period, any male or female that did not show evidence of mating on the day before their FOB assessment, continued to be paired for mating. On the day of testing, the animals were removed from the mating cage and housed singly for approximately 1 hour before testing. After testing, animals were placed back into mating cages as required.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or wood shavings (Datesand Ltd, Manchester, UK).
Each batch of bedding was analysed for specific constituents and contaminants. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
Mains water was provided ad libitum via water bottles (mains water supply). The water is periodically analysed for specific contaminants. No contaminants were present in water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance. The ground diet used for the dietary formulations was Purina Mills Certified Rodent Diet 5002. Each batch of diet was analysed for specific constituents and contaminants. No contaminants were present in diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
The animals were housed in a single, exclusive room. Rooms were air-conditioned to provide 15 to 20 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 20 to 24°C and 45 to 65 % respectively.
Animals were provided with wooden Aspen chew blocks and rodent retreats as forms of environmental enrichment.

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
Dietary formulations were prepared weekly. The test article was formulated as a diet mix in ground diet (Purina Mills Certified Rodent Diet 5002) following Dispensary SOPs and the formulation methods 8304040_d_001D as maintained in the study data.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Administered in the diet to males for 2 weeks prior to pairing, during the pairing period and until the day before necropsy and to the females for 2 weeks prior to pairing, during the pairing period and until Day 4 post-partum, inclusive. The females will be allowed to litter and rear their offspring to Day 4 post-partum. The males will be treated for a minimum of 6 weeks prior to necropsy.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dietary formulations prepared at concentrations of 600 and 30000 ppm were analysed to determine homogeneity and stability. Samples were removed in duplicate from the top, middle and bottom of each formulation. These were analysed for test article concentration to determine homogeneity. The formulations were split into 2 aliquots.
One aliquot was stored at room temperature (15 to 25°C). After 24 hours the formulations were analysed in triplicate to determine stability.
The other aliquot was stored frozen (nominal -20°C). After 12 days and a further 6 hours at room temperature the formulations were analysed in triplicate to determine stability.
This range covered the formulations required for this study.
Samples for homogeneity analysis were also taken from formulations prepared for use on the first and last days of dosing.
Duration of treatment / exposure:
Administered in the diet to males for 2 weeks prior to pairing, during the pairing period and until the day before necropsy and to the females for 2 weeks prior to pairing, during the pairing period and until Day 4 post-partum, inclusive. The females will be allowed to litter and rear their offspring to Day 4 post-partum. The males will be treated for a minimum of 6 weeks prior to necropsy.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 male and 10 females per dose.
Control animals:
yes
yes, concurrent no treatment
Details on study design:
Dose levels are based on the absence of findings at the limit dose in a 14-day dietary range-finding study. The high dose was selected to indicate toxicity or its absence at the limit dose.
The intermediate and low dose levels are geometric increments of the high dose.
A readily available rodent species acceptable to the regulatory authorities and recommended for reproduction studies because of its reproductive characteristics.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
All animals were observed at the beginning and the end of the working day for signs of ill health or overt toxicity.
Each animal was given a detailed physical examination at weekly intervals on days of
body weight recording. An individual record was maintained of the clinical condition of each animal.
The animals were also observed once daily. To avoid unnecessary distress around the time of littering for females, observations were not performed from Day 20 of gestation to Day 4 of lactation (inclusive).
Body weights for males were recorded on Day -7, Day -1, Day 1 and weekly
thereafter, including the day of necropsy.
Body weights for females were recorded weekly from Day -7, Day -1, Day 1 and then weekly until confirmation of mating. Body weights were then recorded on Days 0, 6, 7, 13, 14 and 20 of gestation and Days 0, 1 and 4 of lactation.
Food consumption was recorded daily throughout the study and calculated as g/animal/day.
Food consumption recorded during the pairing period was for welfare reasons only and is not reported.
Sacrifice and pathology:
adrenal W E oviduct E
animal identification1 pancreas E
aorta E Peyer’s patch E
bone marrow smear (femur)2 pituitary E
brain W E prostate1 W E
caecum E rectum E
colon E seminal vesicle1 W E
duodenum E spinal cord, cervical E
femur with bone marrow and
stifle joint
E spinal cord, lumbar E
gross lesions1 E spinal cord, thoracic E
heart W E spleen W E
ileum E sternum with bone marrow E
jejunum E stomach E
kidney W E testis with epididymis1, 3, 5 W4 E
larynx E thymus W E
liver W E thyroid with parathyroid W E
lungs E tongue E
lymph node, mandibular E trachea E
lymph node, mesenteric E trachea bifurcation E
lymph node, popliteal E ureter E
nerve, sciatic E urinary bladder1 E
oesophagus E uterus with cervix1 W E
ovary1 W E vagina1 E

Bone designated for histopathological examination was decalcified using Kristenson’s fluid.
The uterus of all females was immersed in 10 % ammonium sulphide solution and implantation sites counted.
Statistics:
Performed where appropriate using standard Covance methods in Pristima data capture and reporting system, current at time of reporting and detailed in SOP STATS31.
Note: For organ weights, Analysis of Covariance with necropsy body weight as the covariate will be used.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs related to treatment with Lime Oxide.
The clinical signs observed were confined to instances of thin fur, sores/lesions on the skin and pale teeth, and seen in all treatment groups. Such findings are commonly observed in laboratory maintained rats and were considered to be unrelated to
treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight losses were evident for males receiving 1000 mg/kg/day during the
study. This resulted on lower mean body weights when compared with controls from
Day 4 onwards, with statistical significance achieved from Day 11 until pairing on
Day 15 (p<0.05 to p<0.01). Mean body weights were still lower after the pairing
phase until the end of treatment when compared with controls (p<0.001), although
body weight change was comparable to controls. Mean body weight change for the
duration of the study was 59% lower than controls.
Mean body weights for males receiving 300 mg/kg/day were also lower than controls
after pairing, until the end of the study (p<0.05 to p<0.01); overall body weight
change at this level was 22% lower than controls.
No adverse effects on body weight change were evident for males receiving
100 mg/kg/day.
For females receiving 1000 mg/kg/day, initial body weight losses were evident, which
resulted in lower mean body weights from Day 4 onwards (p<0.001), when compared
with controls. Reduced mean body weight was still evident throughout the gestation
and early lactation phase at this dose level (p<0.001), with lower mean body weight
change observed from Days 0 to 13 of gestation (p<0.05 to p<0.01) and Days 1 to 4 of
lactation (p<0.001) when compared with controls. Mean body weight change for the
duration of the study was 70 % lower than controls.
An initial reduction in mean body weight gain was evident for females receiving
300 mg/kg/day on Day 4 of treatment when compared with controls (p<0.05).
Recovery was evident for the remainder of the pre-pairing period. There were no
overt differences in mean body weights during early gestation, however, lower body
weight gain was evident from Gestation Day 13 onwards (p<0.01 to p<0.05) when
compared with controls. Lower mean body weights and body weight change were
also observed during early lactation at this level (p<0.01), when compared with
controls. Mean body weight change for the duration of the study was 32 % lower than
controls.
No adverse effect on body weight change was evident for females receiving
100 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower mean food consumption was observed for all male treatment groups when
compared with controls. Mean food consumption for males receiving 1000 mg/kg/day
was significantly reduced between Days 1 and 4 (-42%) when compared with
controls, correlating with the body weight losses observed. Overall food consumption
was 23% lower than controls during the pre-pairing phase and 29% lower than
controls, after the pairing phase, until the end of treatment.
Males receiving 300 or 100 mg/kg/day, showed slightly lower mean food
consumption when compared with controls over the course of the study (-10%
and -6% respectively).
Females receiving 1000 mg/kg/day showed a 55% lower mean food consumption
during the first four days of treatment when compared with controls, correlating with
the body weight losses observed. Mean food consumption during the two week
pre-pairing phase was 35% lower than controls. Mean food consumption was 35%
lower than controls during gestation, and 47% lower than controls during the early
lactation period.
At 300 mg/kg/day, 18% lower mean food consumption was observed during the
pre-pairing phase, 14% lower during gestation and 16% lower during the early
lactation phase, when compared with control values.
At 100 mg/kg/day, mean food intake was 9% lower than controls during the first two
weeks of treatment. Mean food consumption during gestation was essentially similar
to controls and 21 % more than controls during the early lactation phase.
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Total protein levels were significantly lower for both males and females receiving
1000 mg/kg/day when compared with controls, with the effect also observed for
300 mg/kg/day females (p<0.001). Furthermore, females from the 1000 and
300 mg/kg/day dose groups showed significantly lower albumin levels when
compared their concurrent control (p<0.001).
There were no further changes in the clinical chemistry parameters which were
considered to be related to treatment with Lime Oxide. No Lime Oxide related effects
were detected in the haematology parameters investigated.
Any intergroup variation and statistically significant differences in the haematology
parameters, or any remaining intergroup differences in clinical chemistry parameters
were small, inconsistent between the sexes or showed no dose-related response. These
differences were therefore considered to represent normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Males receiving 1000 mg/kg/day showed statistically significant lower forelimb grip
strength on Day 15, when compared with controls (p<0.01). A similar effect was also
observed for females on Day 4 of lactation, although statistical significance was not
achieved.
The remaining neurobehavioural parameters investigated, including those where
statistically significant differences were observed, were considered to be attributable
to normal biological variation, and were considered not to represent a neurotoxic
effect of the test article.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no organ weight changes considered to be related to administration of
Lime Oxide.
Organ weight and/or organ weight ratio changes, including those that were
statistically significant, were attributed to normal biological variation and were
considered not to be related to administration of test article, as they were either small
in magnitude, not dose-dependent, inconsistent between the sexes, due to normal
inter-animal variability and/or lacked a histopathological correlate.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings considered to be related to administration of
Lime Oxide.
Most tissues were either macroscopically unremarkable or the findings seen were
generally consistent with the usual pattern of findings in animals of this strain and
age.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Males receiving 1000 mg/kg/day showed statistically significant lower forelimb grip
strength on Day 15, when compared with controls (p<0.01). A similar effect was also
observed for females on Day 4 of lactation, although statistical significance was not
achieved.
The remaining neurobehavioural parameters investigated, including those where
statistically significant differences were observed, were considered to be attributable
to normal biological variation, and were considered not to represent a neurotoxic
effect of the test article.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No Observed Adverse Effect level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/d

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, continuous oral (dietary) administration of Lime Oxide at nominal dose levels of 100, 300 and 1000 mg/kg/day, to rats for a period of 55 days, including throughout gestation and into early lactation for females, resulted in toxicity at 1000 mg/kg/day. Effects consisted of reduced food consumption with associated reductions in body weight gain and reduced grip strength. Clinical chemistry analysis of the blood showed lower than expected blood protein and albumin levels.
Microscopic changes in the bone marrow and lungs were also observed.
Effects at 300 mg/kg/day were confined to lower body weight gain, and reduced food consumption, with minor changes in blood albumin and total protocol for females.
The findings observed at this dose level were considered to be not adverse, therefore the No Observed Adverse Effect level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day.
Executive summary:

The findings observed at this dose level were considered to be not adverse, therefore the No Observed Adverse Effect level (NOAEL) for systemic toxicity was considered to be 300 mg/kg/day.