Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: inhalation, other
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-reference
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Jan 2015 - 16 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Specific details on test material used for the study:
Identification: Dimetol
Chemical name (IUPAC): 2,6-dimethylheptan-2-ol
Molecular weight: 144.20
CAS Number: 13254-34-7
EC Number: 236-244-1
Description: Clear colourless liquid
Batch: PE00090447*
Purity/Composition: See Certificate of Analysis
Test substance storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 28 May 2016 (expiry date)

* Batch A diets were used until 15 February 2015. Batch B was used to prepare diets from 04 February onwards; these were frozen until use on 16 February 2015.

All information for Batch B is the same as Batch A except for the following:
Batch: PE00105624
Purity/Composition: 99.4%
Stable under storage conditions until: 14 December 2016 (expiry date) (taken from label)
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
other: dietary
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Diet: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Method: The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. No correction was made for the purity/composition of the test substance.

Frequency of preparation: Diets were prepared weekly. Diets were kept frozen ≤ -15°C until needed and were stored at room temperature thereafter. Stability of the test substance in diet was performed in the method validation study; diets in the range of 500 and 15000 ppm (mg test substance/kg diet) were found to be stable for at least 8 days at room temperature (Project 506530).

Storage conditions: After preparation diets were frozen ≤ -15°C until needed at which the diet used was kept at room temperature in the diet store room in the animal house for a maximum of 8 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion (23 January 2015), according to a validated method (Project 506530). Samples of diets were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability of the test substance in diet for at least 8 days at room temperature in the range of 500 and 15000 ppm (mg test substance/kg diet) was confirmed in the method validation study (Project 506530) and was not tested again in the present study.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Random samples were taken from all Groups and stored at ≤-15°C for possible future analysis. Any remaining samples will be discarded at finalization of the study report.
Duration of treatment / exposure:
The males and females were exposed for at least 14 days prior to mating, during mating, and up to the day prior to scheduled necropsy. Males were exposed for 29 days and females were exposed for 39-57 days.
Frequency of treatment:
Oral, by inclusion in the diet - Ad libitum
Remarks:
Doses / Concentrations:
100, 3000 and 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study
Observations and examinations performed and frequency:
Parental animals

Mortality / Viability: At least twice daily.
Clinical signs: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was lso performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4.

Functional Observations The following tests were performed on the selected 5 animals/sex/group:
- hearing ability, pupillary reflex and static righting reflex (Score 0 = normal/present, score 1 = abnormal/absent).
- fore- and hind-limb grip strength were recorded as the mean of three measurements (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum
and on Days 1 and 4 of lactation.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy. Pregnant females were examined to detect signs of difficult or
prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Pups

Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on Day 1 of lactation and daily thereafter. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Sacrifice and pathology:
Pathomorphologic examination was performed on 80 Wistar (Han) rats (40 males, 40 females) which had been subjected to a combined 28 day oral (diet) toxicity study and reproduction/developmental toxicity screening test with the test item Dimetol.
The rats were assigned to four dose groups, each containing 10 males and 10 females. The test item was administered once daily by dietary inclusion at doses of 1000, 3000 and 10000 ppm (dose Groups 2, 3 and 4 respectively). The rats of the control Group 1 received standard powder rodent diet without test item.
The males and females were exposed from 2 weeks prior to mating, during mating, and up to the day prior to necropsy for at least 28 days. All rats were necropsied. Histopathologic examination was performed on an extensive list of organs and tissues from 5 selected males and females of Group 1 and Group 4, heart and kidneys from selected Group 2 and 3 males, spleen from selected Group 2 and 3 females, reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups as well as all organs with macroscopic findings from all rats.
Additional slides of the testes were made of the 5 selected males of Groups 1 and 4 and all males suspected to be infertile and stained with PAS/haematoxylin to assess spermatogenesis.
There were no test item related unscheduled deaths.
Complete postmortem examinations were performed on all animals. Animals were anesthetized using isoflurane and subsequently exsanguinated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 3; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 4) was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 5) was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data(scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
[Note: references can be found in the complete study report]
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Parental animals
There were no adverse clinical signs with treatment up to 10000 ppm. The only clinical sign noted was piloerection, seen for a single day for one female (no. 51) at 1000 ppm. As it was transient and seen for a single low dose female, it was considered incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Parental animals
One control female (no. 47) and one female at 1000 ppm (no. 55) were euthanized before their scheduled necropsies after having total litter losses. Both of these females had only one pup. These were not attributable to treatment and there were no other unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Parental animals
Body weight gains were significantly lower for males at 10000 ppm from Day 8 of the premating period and continuing through the duration of treatment. Body weight gains were lower for females at 3000 and 10000 on Day 8 of the premating period only (not statistically significant).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Parental animals
There were no toxicologically relevant effects on food consumption with treatment up to 10000 ppm. Animals at 10000 ppm (both sexes) had lower absolute and relative food consumption during the first week in treatment. Relative food consumption was lower for females at 3000 ppm during this time. This is likely secondary to palatability issues as food consumption recovered to levels comparable to controls thereafter. Food consumption was higher for all treated males during the first week of the
mating period. This was secondary to slightly low values obtained for one cage of control males and was not considered to be treatment related. Absolute and relative food consumption was significantly higher Days 7-11 of the post coitum period
for females at 1000 ppm. In the absence of a dose-dependent distribution, it was not considered to be toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Parental animals
There were no toxicologically relevant effects on haematology parameters seen up to 10000 ppm. Statistically significant decreases in red blood cell count, haemoglobin, and haematocrit were noted for females at 1000 and 3000 ppm and statistically significant increases were seen in red blood cell distribution width (RDW) and platelet counts for females at 1000 ppm. Significantly lower mean corpuscular haemoglobin concentration (MCHC) was noted for males at 1000 and 3000 ppm. The
statistically significant changes observed at 1000 and 3000 ppm were not toxicologically relevant as the values remained within the range considered normal for this age and strain and/or no dose dependent effect was observed.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Parental animals
There were no toxicologically relevant effects on clinical biochemistry parameters with treatment up to 10000 ppm.
At 10000 ppm potassium was significantly higher for males and bile acids were significantly higher for females. As these values remained within the range of available historical control data, and in the case of bile acids, were considered secondary to slightly low values obtained for controls, they were not considered to be adverse (HCD male potassium mean=4.09±0.245, p95=4.48; female bile acids mean=31.3±19.16). The statistically significant increase in bile acids seen for males at 1000 ppm was not considered to be toxicologically relevant as this occurred in the absence of a dose-dependent distribution.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant effects on functional observations.
At 10000 ppm, high dose males had significantly reduced hind limb grip strength, while forelimb grip strength was significantly reduced for females at this dose level. These values remained within the historical control data available for this parameter and were not accompanied by any clinical signs indicating muscular weakness (HCD mean males: 714±161, p5=387; females: 846±287). Taken together, the differences from controls were not considered to be toxicologically relevant.
Hearing ability, pupil reflex and static righting reflex were normal for all animals tested. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Parental Animals
At 10000 ppm absolute and relative liver weights were increased for animals of both sexes (absolute liver weights for males were not statistically significant). Relative liver weights were approximately 16% and 20% higher for males and females than controls, respectively. The statistically significant increases in ovary weights for animals at 1000 and 3000 ppm and the increase in uterine weights at 1000 ppm occurred in the absence of a dose response and were not considered to be toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on macroscopic findings noted up to 10000 ppm.
Incidental findings seen for control and/or treated animals included isolated or several reddish foci on the lungs, dark red or reddish discoloration of the mandibular lymph node, abnormal shape of the sternum, enlarged thyroid glands, yellowish, hard nodule on the epididymis, reduced size of the seminal vesicle, flaccid kidney and tan focus on the clitoral gland. These findings are occasionally seen for animals of this age and strain and in the absence of a treatment related distribution, they were not considered to be toxicologically relevant. An early resorption in the right uterine horn was noted for the control female with a total litter loss.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were present in males in the kidney where hyaline droplet accumulation was present at increased incidence and severity in males treated at 10000 ppm up to a moderate degree. Tubular basophilia was present at an increased incidence and severity in males treated at 10000 ppm up to a slight degree. Granular casts were present in a single male treated at 10000 ppm at slight degree. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined. In female Wistar Han rats, there were no test item-related morphologic alterations after treatment with Dimetol for at least 28 days at a dose up to 10000 ppm.
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: parental No Observed Adverse Effect Level (NOAEL)
Critical effects observed:
no

Accuracy and homogeneity of diets were demonstrated by analyses.

Parental results:

Parental toxicity was evident at 10000 ppm and was characterized by lower body weight gains for males Day 8 of the premating period onwards. Palatability issues of the test diet likely contributed to

the lower gains as food consumption was lower during the first week of treatment. Males and females at this dose level had higher absolute and relative liver weights with relative liver weights approximately 16% and 20% higher for males and females than controls, respectively. At the microscopic examination, cortical hyaline droplets representing alpha2uglobulin were recorded at an increased incidence and severity in the kidneys of males treated at 10000 ppm. These changes resulted from the increased/altered liver metabolism resulting in excessive accumulation of alpha- 2uglobulin in the renal proximal tubular epithelial cells and are regarded as specific to the male rat. Alpha-2uglobulin does not occur in the human kidney and that this finding does not indicate any potential risk to human health. This was companied by an increased incidence and severity of corticomedullary tubular basophilia and in one instance by granular casts. These granular casts are considered to be indicative of primary tubular injury and therefore this finding was considered to be adverse.

There were no relevant microscopic findings noted for females at 10000 ppm.

No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, clinical laboratory investigations and macroscopic examination).

Conclusions:
In conclusion, treatment with Dimetol by dietary administration in male and female Wistar Han rats at dose levels of 1000, 3000 and 10000 ppm revealed parental toxicity at 10000 ppm. No reproductive or developmental toxicity was observed for treatment up to 10000 ppm.
Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 3000 ppm was determined.
When corrected for mean test article intake, the parental NOAEL of 3000 ppm corresponds to 228-231 mg/kg for males and 251-382 mg/kg for females.
Executive summary:

Dimetol was administered by diet to male and female Wistar Han rats at dose levels of 1000, 3000 and 10000 ppm. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during postcoitum, and at least 4 days of lactation (for 39-57 days).

There were no relevant microscopic findings noted for females at 10000 ppm. No treatment-related changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, clinical laboratory investigations and macroscopic examination). Reproductive results: No reproductive toxicity was observed up to the highest dose level tested (10000 ppm). Developmental results: No developmental toxicity was observed up to the highest dose level tested (10000 ppm)

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 3000 ppm was determined and a reproductive and developmental NOAEL of at least 10000 ppm was derived. When corrected for mean test article intake, the parental NOAEL of 3000 ppm corresponds to 228-231 mg/kg for males and 251-382 mg/kg for females. The reproductive and developmental NOAEL of 10000 ppm corresponds to 714-734 mg/kg for males and 830-1216 mg/kg for females..

Data source

Materials and methods

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion