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Description of key information

Extended-Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test: NOAEL = 700 ppm (based on reduced food consumption of females throughout the post-coitum and lactation period at 2000 ppm (approximately 30-40% lower than controls), hypertrophy of the urothelium of the urinary bladder in females at 2000 ppm and renal changes in males at 2000 ppm (hyaline droplet accumulation, accompanied by renal tubular basophilia and granular casts and associated higher renal weight).

[700 ppm in the diet corresponded to mean daily test item intake levels of 51 mg/kg bw/day in males, and 59 (pre-mating period), 87 (post-coitum period) and 129 (lactation period) mg/kg bw/day in females.]

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:
key study
Study period:
14 October 2015 to 11 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 422 without any deviation.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Mars 1996
Deviations:
yes
Remarks:
exposure period was longer than a standard OECD 422 (males were exposed for 91 days, and females for 103-114 days)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870-3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch B.V. has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: Yes
- Age at start F0-treatment: Approximately 6 weeks
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: Free access to prepared diets. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Tap-water, ad libitum
- Acclimation period: 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 18-24°C
- Humidity: 40-70 %
- Air changes: 10 room air changes/hour
- Photoperiod: 12-hour light/12-hour dark

IN-LIFE DATES: 14 October 2015 to 11 March 2016
Route of administration:
oral: feed
Details on route of administration:
The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Diet: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Method: The test item was mixed without the use of a vehicle (w/w), directly with the required amount of powder feed. No correction was made for the purity or specific gravity of the test item.
- Frequency of preparation: Diets were prepared up to 3 weeks in advance of first use.
- Storage of preparations: Diets were kept in the freezer (≤-15 °C) until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 8 days for supplementing food during the respective food consumption measurement interval.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on diet samples taken on a single occasion during the treatment phase (17 November 2015), according to a validated method (Test Facility Study No. 509508). Samples of diet preparations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in diet over 8 days at room temperature and 3 weeks in the freezer were also determined for 250 ppm diets.
Stability in rat diet (powder, SM R/M-Z; supplier SSNIFF®) over the concentration range 500 to 15,000 ppm was previously confirmed for at least 3 weeks in the freezer (≤-15 °C) and for at least 8 days at room temperature (Project 509508 (method development and validation 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%. Diet preparations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 91 days, i.e. 10 weeks prior to mating, during mating, and up to termination.
Females were exposed for 103 - 114 days, i.e. during 10 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy for selected females and until the day of necropsy for non-selected females).
Frequency of treatment:
ad libitum
Dose / conc.:
0 ppm
Remarks:
Group 1
Dose / conc.:
250 ppm
Remarks:
Group 2
Dose / conc.:
700 ppm
Remarks:
Group 3
Dose / conc.:
2 000 ppm
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/dose
5 animals/sex/group were selected for functional observations, locomotor activity, clinical pathology, macroscopic examination, organ weights and histopathology
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 14-day dose range finding study (Study No. 509507). In this study, rats (3/sex/dose) were administered test substance at the dose levels of 0, 500, 1500 and 5000 ppm in the diet for 14 days. No mortality was observed. Hunched posture (one female between Days 12-14) was observed at 5000 ppm. At 5000 ppm, reduced weight gain for one male; slight weight loss for two females between Days 1-8 (up to 3% from Day 1 values) were observed. No change in food consumption was observed. Test item-related morphologic alterations following the administration of test substance for 14 days by diet were present in the kidneys of male rats only. These findings consisted of a dose-related increase in incidence and severity of hyaline droplet accumulation up to marked degree. This hyaline droplet accumulation is most likely representing alpha2uglobulin, a normal protein in male rats, not present in female rats or in higher mammals, including man. From 1500 ppm onwards, this accumulation was accompanied by tubular degeneration (up to moderate) and at 5000 ppm also by the presence of granular casts up to moderate degree (tubular dilation filled by degenerated cells). Based on the degenerative character, the kidney findings at 1500 and 5000 ppm were considered to be adverse for the male rats, but is presumably a species and sex specific phenomena. There were no target organs identified in the female rats treated up to doses of 5000 ppm. Based on the results of this range finding study, dose levels for the main study were: 250, 700 and 2000 ppm.
- Rationale for animal assignment (F0): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule:
Mortality / Viability: At least twice daily.
Clinical signs: At least once daily from start of 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 also performed outside the home cage in a standard arena. When animals were littering at the time of conducting arena observations, there were not subjected to these observations.

BODY WEIGHT: Yes
- Time schedule for examinations: 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 AND COMPOUND INTAKE (if feeding study): Yes
- 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 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group.
- Anaesthetic used for blood collection: Yes; animals were held under anaesthesia using isoflurane
- Animals fasted: Yes; animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
- Parameters checked:
Haematology: White blood cells (WBC), Differential leucocyte count: (Neutrophils, Lymphocytes, Monocytes, Eosinophils & Basophils), Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC) and Platelets
Clotting parameters: Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)
Clinical chemistry: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium and Inorganic Phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 animals/sex/group; The selected males were tested during Week 13 of treatment and the selected females were tested towards the end of the scheduled lactation period from lactation Day 4 onwards (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, when applicable).
- Battery of functions tested: Hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R); fore and hindlimb grip strength; locomotor activity

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
- All males and the selected 5 females/group were deprived of food overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
- Females which delivered: Necropsy was performed on Lactation Days 5-7.
- Females which failed to deliver (nos. 54, 67, 69 and 74): Post-coitum Days 25-26 (females with evidence of mating)
- Females with total litter loss (no. 55): Within 24 hours of litter loss.
- Males: Following completion of the mating period (after 91 days of dose administration).

GROSS NECROPSY
- Animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ Weights: Terminal body weights were recorded from all males and the selected 5 females/sex/group. The following organ weights were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group: Adrenal glands, Brain -cerebellum, midbrain, cortex (7 levels), Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Thymus, Testes, Thyroid including parathyroid and Uterus (including cervix)
All remaining males: Epididymides and Testes
Histopathology: The tissues indicated in Table 7.8.1/1 were prepared for microscopic examination. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands). All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile or which died before mating to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The kidneys of the selected 5 males of Groups 2 and 3, the liver and thyroid of the selected 5 males and females of Groups 2 and 3, and the urinary bladder and thymus of the selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- The reproductive organs* of all males that failed to sire and all females that failed to deliver healthy pups:
Group 2 female/male no. 54/14 (not pregnant); 55/15 (total litter loss)
Group 3 female/male no. 67/27 (not pregnant); 69/29 (not pregnant)
Group 4 female/male no. 74/34 (not pregnant)
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
Other examinations:
None
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 (many-to-one 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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test 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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No clinical signs or abnormalities during weekly arena observations were noted that were considered to be related to treatment.
- The only clinical finding noted consisted of scabs on the back in a single female at 250 ppm. This incidental finding was not related to treatment and within the range of background findings expected for rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
- No mortality occurred during the study period.
- One female at 250 ppm (no. 55) was sacrificed due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Pre-mating body weight gain of females at 2000 ppm was slightly lower compared to controls (percentual difference in mean body weight gain was about 10% in most weeks, being statistically significant at Days 50 and 71).
- Throughout the post-coitum and lactation periods, mean body weights of females at 2000 ppm were statistically significantly lower compared to controls (relative differences from controls were about 10%). However, body weight gain values during the post-coitum and lactation periods at 2000 ppm were considered similar to controls.
- No treatment-related changes in body weights or body weight gain were noted up to 2000 ppm in males and up to 700 ppm in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Throughout the post-coitum and lactation period, females at 2000 ppm had a notably lower food intake than controls (approximately 30-40% lower than controls). Food consumption after allowance for body weight showed a similar pattern, but was not statistically significantly lower during lactation. During premating, food consumption before and after allowance for body weight was similar to control levels.
- No treatment-related changes in food consumption before or after allowance for body weight were noted up to 2000 ppm in males and up to 700 ppm in females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters of treated rats were not affected by treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
- Statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the change (values in treated rats remained within normal limits), low control values and/or absence of a dose-related response.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
- Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity 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):
- Test item-related higher liver weights were noted in the 2000 ppm treated males (absolute and relative to body weight). Mean relative weight was approximately 13% higher than controls.
- Test item-related higher kidneys weights were noted in the 700 and 2000 ppm treated males (relative to body weight at 700 ppm and both absolute and relative to body weight at 2000 ppm). Mean relative weight was approximately 13% and 15% higher than controls at 700 and 2000 ppm, respectively.
- Test item-related higher kidneys weights were also noted in the 250, 700 and 2000 ppm treated females (relative to body weight at 250 and 2000 ppm and both absolute and relative to body weight at 700 ppm). Mean relative weights were approximately 13%, 21% and 16% higher than controls at 250, 700 and 2000 ppm, respectively. No dose-related trend was apparent.
- There were no other test item-related organ weight changes. All other organ weight differences observed, including those that reached statistical significance, were considered incidental and unrelated to the administration of the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Enlargement of the liver was noted in 2/10 males at 2000 ppm. This macroscopic finding was considered to be test item-related.
- The remainder of the recorded macroscopic findings were within the range of background findings encountered in rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: hepatocellular hypertrophy in 4/5 males and 4/5 females at 2000 ppm up to slight degree.
- Kidneys: increased incidence and severity of hyaline droplet accumulation in males at 2000 ppm up to moderate degree, accompanied by an increased incidence and severity of tubular basophilia up to slight degree at 2000 ppm and tubular granular casts in a single male treated at 2000 ppm at minimal degree.
- Urinary bladder: Hypertrophy of the urothelium in females at 2000 ppm up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
700 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
food consumption and compound intake
histopathology: non-neoplastic
Remarks on result:
other: 700 ppm in the diet corresponded to mean daily test item intake levels of 59 (pre-mating period), 87 (post-coitum period) and 129 (lactation period) mg/kg bw/day in females
Key result
Dose descriptor:
NOAEL
Effect level:
700 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: 700 ppm in the diet corresponded to mean daily test item intake levels of 51 mg/kg bw/day in males
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Analysis of Diet Preparations

Accuracy of preparation: The concentrations analysed in the diets at 250, 700 and 2000 ppm were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

No test item was detected in the Group 1 diets.

Homogeneity: The diets at 250 and 2000 ppm were homogeneous (i.e. coefficient of variation ≤10%).

Stability: Diets at 250 ppm were stable when stored at room temperature under normal laboratory light conditions in an open container for at least 8 days (i.e. difference between the stored and freshly taken samples was 10%). The relative difference of diet samples at low level 250 ppm for 3 weeks in the freezer (≤-15°C) stability test was slightly above the criterion (i.e. - 13%). The decrease was considered not to be due to instability of the samples since the mean accuracy of the stability samples was comparable to the accuracy of the quality control samples at the same concentration level which were analysed together with the 3-week stability samples. Furthermore, since the deviations were considered small and accuracies after storage were within the range of 80 and 120%, the diets were considered stable. Based on this, the diets from 250 ppm to 15000 ppm (project 509508) were found to be stable during storage at room temperature under normal laboratory light conditions with open container for at least 8 days and in a freezer (≤-15°C) for at least 3 weeks.

 

Table 7.8.1/2: Food Test Article Intake

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table below.

 

Males / Females

Test item intake (mg test item/kg bw/day)1

Group 2

250 ppm

Group 3

700 ppm

Group 4

2000 ppm

Males

Pre-mating + Mating

19

51

150

Females

Pre-mating

21

59

168

Post-coitum

32

87

184

Lactation

44

129

280

1Values are the overall group means in the periods indicated.

Conclusions:
Under the test conditions, the parental NOAEL in male and female rats was 700 ppm (based on reduced food consumption of females throughout the post-coitum and lactation period at 2000 ppm (approximately 30-40% lower than controls), hypertrophy of the urothelium of the urinary bladder in females at 2000 ppm and renal changes in males at 2000 ppm (hyaline droplet accumulation, accompanied by renal tubular basophilia and granular casts and associated higher renal weight).
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, groups of Crl:WI(Han) rats (10/sex/dose) received test substance in the diet, at concentrations of 0, 250, 700 and 2000 ppm.Males were exposed for 91 days, i.e. 10 weeks prior to mating, during mating, and up to termination. Females were exposed for 103 - 114 days, i.e. during 10 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

 

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues.

 

The concentrations analysed in the diets at 250, 700 and 2000 ppm were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

 

There were no treatment-related changes in mortality, clinical appearance, and functional observations up to 2000 ppm.

 

There were no adverse changes in in-life parameters (body weight, food consumption, clinical signs and functional observations) across the dose groups. A lower body weight and food consumption was recorded for females treated at 2000 ppm throughout the post-coitum and lactation periods, and for body weight also during the premating period. Compared to controls, mean body weights were about 10% lower than controls, whereas the differences in food consumption were more marked (approximately 30-40% lower than controls throughout the post-coitum and lactation period). This lower food intake was however not considered adverse in nature since it occurred in absence of any adverse changes in body weight or clinical appearance, or treatment-related changes in pup body weights.

 

No toxicologically relevant changes were noted in clinical biochemistry or haematological parameters up to 2000 ppm.

 

Microscopic examination revealed minimal to slight hypertrophy of the urothelium in the urinary bladder of females treated at 2000 ppm. This test item-related change was considered to be adverse in this > 90-day study because it was seen in three out of five females, none in the control and it is an effect which does not typically occur as background finding. It does not seem to be a secondary response to irritation by calculi, lower urinary tract obstruction or in association with renal papillary necrosis as these effects were not observed. Therefore the bladder effects are possibly a primary effect of the test item. The mode of action for this effect is unclear and therefore the effect is used for deriving the NOAEL. There are, however, no indications that the kidney function in females was impaired based on other kidney (related) findings. In addition, there were no related findings seen such as morphological lesions (e.g. inflammation or cell death) and it was not seen in males.

 

Additionally, an adverse histopathological lesion was recorded in the kidneys of males treated at 2000 ppm. In these males, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted. The hyaline droplet accumulation was considered to represent alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man. In this study the increased hyaline droplet accumulation in males treated at 2000 ppm was accompanied by indicators of renal tubular damage in the form of granular casts at minimal degree and increased tubular basophilia up to slight degree. This combination of findings was considered to be adverse. In addition, males at 2000 ppm had kidney weights that were approximately 15% higher than controls and were considered to be related to the microscopic renal changes. Higher kidney weights were also recorded for males at 700 ppm (approximately 13% higher than controls), and for females at 250, 700 and 2000 ppm (approximately 13%, 21% and 16% higher, respectively). There were neither corroborative microscopic changes nor clinical biochemical alterations indicative of renal toxicity. Furthermore, kidney weights remained within the range considered normal for this strain of rats of similar age. Therefore, the higher kidney weights for males at 700 ppm and for females at 250 ppm and higher were not considered adverse in nature.

 

A treatment-related but non-adverse hepatocellular hypertrophy was recorded in the liver of males and females treated at 2000 ppm up to slight degree. This microscopic finding correlated with a modest increase in liver weight (relative weight approximately 13% higher than controls) and macroscopic liver enlargement observed in a few 2000 ppm males. In the absence of any other indicators of hepatocellular toxicity, these liver changes were not considered to be adverse.

 

Under the test conditions, the parental NOAEL in male and female rats was 700 ppm (based on reduced food consumption of females throughout the post-coitum and lactation period at 2000 ppm (approximately 30-40% lower than controls), hypertrophy of the urothelium of the urinary bladder in females at 2000 ppm and renal changes in males at 2000 ppm (hyaline droplet accumulation, accompanied by renal tubular basophilia and granular casts and associated higher renal weight).

NB:

700 ppm in the diet corresponded to mean daily test item intake levels of 51 mg/kg bw/day in males, and 59 (pre-mating period), 87 (post-coitum period) and 129 (lactation period) mg/kg bw/day in females.

2 2000 ppm in the diet corresponded to mean daily test item intake levels of 150 mg/kg bw/day in males, and 168 (pre-mating period), 184 (post-coitum period) and 280 (lactation period) mg/kg bw/day in females.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, (eco)toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are 1-[(x,x)-dimethyl-1-cyclohexen-1-yl]-pent-4-en-1-one, which can exist as alpha (1-[(5,5)-dimethyl...) or beta (1-[(3,3)-dimethyl...) forms, meaning the position of the double bond in the hexane cycle differs.
The target substance is the isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one).
The source substance is a mixture of isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present as the major constituent between 60 and 75% in the mixture, and corresponding to the target (mono-constituent) substance; and isomer beta (1-(3,3-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present between 25 and 35%.

3. ANALOGUE APPROACH JUSTIFICATION
The source substance is expected to have similar toxicological profile than the target substance considering the respective physico-chemical and toxicological properties.
The study design (OECD 422, GLP) is adequate and reliable for the purpose of the prediction based on read-across. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the sub-chronic toxicity studies conducted in the rat with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VIII, 8.6.2.

4. DATA MATRIX
See Iuclid section13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- No clinical signs or abnormalities during weekly arena observations were noted that were considered to be related to treatment.
- The only clinical finding noted consisted of scabs on the back in a single female at 250 ppm. This incidental finding was not related to treatment and within the range of background findings expected for rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
- No mortality occurred during the study period.
- One female at 250 ppm (no. 55) was sacrificed due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Pre-mating body weight gain of females at 2000 ppm was slightly lower compared to controls (percentual difference in mean body weight gain was about 10% in most weeks, being statistically significant at Days 50 and 71).
- Throughout the post-coitum and lactation periods, mean body weights of females at 2000 ppm were statistically significantly lower compared to controls (relative differences from controls were about 10%). However, body weight gain values during the post-coitum and lactation periods at 2000 ppm were considered similar to controls.
- No treatment-related changes in body weights or body weight gain were noted up to 2000 ppm in males and up to 700 ppm in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Throughout the post-coitum and lactation period, females at 2000 ppm had a notably lower food intake than controls (approximately 30-40% lower than controls). Food consumption after allowance for body weight showed a similar pattern, but was not statistically significantly lower during lactation. During premating, food consumption before and after allowance for body weight was similar to control levels.
- No treatment-related changes in food consumption before or after allowance for body weight were noted up to 2000 ppm in males and up to 700 ppm in females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters of treated rats were not affected by treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
- No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
- Statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the change (values in treated rats remained within normal limits), low control values and/or absence of a dose-related response.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
- Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity 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):
- Test item-related higher liver weights were noted in the 2000 ppm treated males (absolute and relative to body weight). Mean relative weight was approximately 13% higher than controls.
- Test item-related higher kidneys weights were noted in the 700 and 2000 ppm treated males (relative to body weight at 700 ppm and both absolute and relative to body weight at 2000 ppm). Mean relative weight was approximately 13% and 15% higher than controls at 700 and 2000 ppm, respectively.
- Test item-related higher kidneys weights were also noted in the 250, 700 and 2000 ppm treated females (relative to body weight at 250 and 2000 ppm and both absolute and relative to body weight at 700 ppm). Mean relative weights were approximately 13%, 21% and 16% higher than controls at 250, 700 and 2000 ppm, respectively. No dose-related trend was apparent.
- There were no other test item-related organ weight changes. All other organ weight differences observed, including those that reached statistical significance, were considered incidental and unrelated to the administration of the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Enlargement of the liver was noted in 2/10 males at 2000 ppm. This macroscopic finding was considered to be test item-related.
- The remainder of the recorded macroscopic findings were within the range of background findings encountered in rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: hepatocellular hypertrophy in 4/5 males and 4/5 females at 2000 ppm up to slight degree.
- Kidneys: increased incidence and severity of hyaline droplet accumulation in males at 2000 ppm up to moderate degree, accompanied by an increased incidence and severity of tubular basophilia up to slight degree at 2000 ppm and tubular granular casts in a single male treated at 2000 ppm at minimal degree.
- Urinary bladder: Hypertrophy of the urothelium in females at 2000 ppm up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
700 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
food consumption and compound intake
histopathology: non-neoplastic
Remarks on result:
other: 700 ppm in the diet corresponded to mean daily test item intake levels of 59 (pre-mating period), 87 (post-coitum period) and 129 (lactation period) mg/kg bw/day in females
Key result
Dose descriptor:
NOAEL
Effect level:
700 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: 700 ppm in the diet corresponded to mean daily test item intake levels of 51 mg/kg bw/day in males
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 000 ppm
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Analysis of Diet Preparations

Accuracy of preparation: The concentrations analysed in the diets at 250, 700 and 2000 ppm were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

No test item was detected in the Group 1 diets.

Homogeneity: The diets at 250 and 2000 ppm were homogeneous (i.e. coefficient of variation ≤10%).

Stability: Diets at 250 ppm were stable when stored at room temperature under normal laboratory light conditions in an open container for at least 8 days (i.e. difference between the stored and freshly taken samples was 10%). The relative difference of diet samples at low level 250 ppm for 3 weeks in the freezer (≤-15°C) stability test was slightly above the criterion (i.e. - 13%). The decrease was considered not to be due to instability of the samples since the mean accuracy of the stability samples was comparable to the accuracy of the quality control samples at the same concentration level which were analysed together with the 3-week stability samples. Furthermore, since the deviations were considered small and accuracies after storage were within the range of 80 and 120%, the diets were considered stable. Based on this, the diets from 250 ppm to 15000 ppm (project 509508) were found to be stable during storage at room temperature under normal laboratory light conditions with open container for at least 8 days and in a freezer (≤-15°C) for at least 3 weeks.

 

Table 7.8.1/2: Food Test Article Intake

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table below.

 

Males / Females

Test item intake (mg test item/kg bw/day)1

Group 2

250 ppm

Group 3

700 ppm

Group 4

2000 ppm

Males

Pre-mating + Mating

19

51

150

Females

Pre-mating

21

59

168

Post-coitum

32

87

184

Lactation

44

129

280

1Values are the overall group means in the periods indicated.

Conclusions:
Under the test conditions, the parental NOAEL for the source substance in male and female rats was 700 ppm (based on reduced food consumption of females throughout the post-coitum and lactation period at 2000 ppm (approximately 30-40% lower than controls), hypertrophy of the urothelium of the urinary bladder in females at 2000 ppm and renal changes in males at 2000 ppm (hyaline droplet accumulation, accompanied by renal tubular basophilia and granular casts and associated higher renal weight).
Based on the results on the source substance, the NOAEL for the target substance is set to 700 ppm (the source and the target substance having the same molecular weight, the correction for molecular weight is not needed).
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, groups of Crl:WI(Han) rats (10/sex/dose) received the source substance in the diet, at concentrations of 0, 250, 700 and 2000 ppm.Males were exposed for 91 days, i.e. 10 weeks prior to mating, during mating, and up to termination. Females were exposed for 103 - 114 days, i.e. during 10 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

 

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues.

 

The concentrations analysed in the diets at 250, 700 and 2000 ppm were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

 

There were no treatment-related changes in mortality, clinical appearance, and functional observations up to 2000 ppm.

 

There were no adverse changes in in-life parameters (body weight, food consumption, clinical signs and functional observations) across the dose groups. A lower body weight and food consumption was recorded for females treated at 2000 ppm throughout the post-coitum and lactation periods, and for body weight also during the premating period. Compared to controls, mean body weights were about 10% lower than controls, whereas the differences in food consumption were more marked (approximately 30-40% lower than controls throughout the post-coitum and lactation period). This lower food intake was however not considered adverse in nature since it occurred in absence of any adverse changes in body weight or clinical appearance, or treatment-related changes in pup body weights.

 

No toxicologically relevant changes were noted in clinical biochemistry or haematological parameters up to 2000 ppm.

 

Microscopic examination revealed minimal to slight hypertrophy of the urothelium in the urinary bladder of females treated at 2000 ppm. This test item-related change was considered to be adverse in this > 90-day study because it was seen in three out of five females, none in the control and it is an effect which does not typically occur as background finding. It does not seem to be a secondary response to irritation by calculi, lower urinary tract obstruction or in association with renal papillary necrosis as these effects were not observed. Therefore the bladder effects are possibly a primary effect of the test item. The mode of action for this effect is unclear and therefore the effect is used for deriving the NOAEL. There are, however, no indications that the kidney function in females was impaired based on other kidney (related) findings. In addition, there were no related findings seen such as morphological lesions (e.g. inflammation or cell death) and it was not seen in males.

 

Additionally, an adverse histopathological lesion was recorded in the kidneys of males treated at 2000 ppm. In these males, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted. The hyaline droplet accumulation was considered to represent alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man. In this study the increased hyaline droplet accumulation in males treated at 2000 ppm was accompanied by indicators of renal tubular damage in the form of granular casts at minimal degree and increased tubular basophilia up to slight degree. This combination of findings was considered to be adverse. In addition, males at 2000 ppm had kidney weights that were approximately 15% higher than controls and were considered to be related to the microscopic renal changes. Higher kidney weights were also recorded for males at 700 ppm (approximately 13% higher than controls), and for females at 250, 700 and 2000 ppm (approximately 13%, 21% and 16% higher, respectively). There were neither corroborative microscopic changes nor clinical biochemical alterations indicative of renal toxicity. Furthermore, kidney weights remained within the range considered normal for this strain of rats of similar age. Therefore, the higher kidney weights for males at 700 ppm and for females at 250 ppm and higher were not considered adverse in nature.

 

A treatment-related but non-adverse hepatocellular hypertrophy was recorded in the liver of males and females treated at 2000 ppm up to slight degree. This microscopic finding correlated with a modest increase in liver weight (relative weight approximately 13% higher than controls) and macroscopic liver enlargement observed in a few 2000 ppm males. In the absence of any other indicators of hepatocellular toxicity, these liver changes were not considered to be adverse.

 

Under the test conditions, the parental NOAEL for the source substance in male and female rats was 700 ppm (based on reduced food consumption of females throughout the post-coitum and lactation period at 2000 ppm (approximately 30-40% lower than controls), hypertrophy of the urothelium of the urinary bladder in females at 2000 ppm and renal changes in males at 2000 ppm (hyaline droplet accumulation, accompanied by renal tubular basophilia and granular casts and associated higher renal weight).

NB:

700 ppm in the diet corresponded to mean daily test item intake levels of 51 mg/kg bw/day in males, and 59 (pre-mating period), 87 (post-coitum period) and 129 (lactation period) mg/kg bw/day in females.

2 2000 ppm in the diet corresponded to mean daily test item intake levels of 150 mg/kg bw/day in males, and 168 (pre-mating period), 184 (post-coitum period) and 280 (lactation period) mg/kg bw/day in females.

Based on the results on the source substance, the NOAEL for the target substance is set to 700 ppm (the source and the target substance having the same molecular weight, the correction for molecular weight is not needed).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
51 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
No study was located on the target substance. The study on the source substance is GLP-compliant and of high quality (Klimisch score = 1).
System:
urinary
Organ:
bladder
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No study was located on the target substance. The study on the source substance (Charles River, 2016, rel.1) is the key study for this endpoint (see Iuclid section 13 for read-across justification).

In this Extended-Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test performed according to OECD test guideline No. 422 and in compliance with GLP, the source substancewas administered via the diet to male and female Wistar Han rats at dietary concentrations of 250, 700 and 2000 ppm (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received basal diet without test item. Males were exposed for 10 weeks prior to mating, during mating, and up to termination (for 91 days). The females were exposed for 10 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 103 - 114 days).

Analysis of diet preparations showed that the diets were prepared accurately and homogenously, and were stable during storage at room temperature under normal laboratory light conditions in an open container for at least 8 days and in a freezer (≤ -15°C) for at least 3 weeks.

There were no adverse changes in in-life parameters (body weight, food consumption, clinical signs and functional observations) across the dose groups. A lower body weight and food consumption was recorded for females treated at 2000 ppm throughout the post-coitum and lactation periods, and for body weight also during the premating period. Compared to controls, mean body weights were about 10% lower than controls, whereas the differences in food consumption were more marked (approximately 30-40% lower than controls throughout the post-coitum and lactation period). This lower food intake was however not considered adverse in nature since it occurred in absence of any adverse changes in body weight or clinical appearance, or treatment-related changes in pup body weights.

There were no treatment-related changes in mortality, clinical appearance, and functional observations up to 2000 ppm. There were no treatment-related changes in other in-life parameters including mortality, clinical appearance, and functional observations up to 2000 ppm.

Microscopic examination revealed minimal to slight hypertrophy of the urothelium in the urinary bladder of females treated at 2000 ppm. This test item-related change was considered to be adverse in this > 90-day study because it was seen in three out of five females, none in the control and it is an effect which does not typically occur as background finding. It does not seem to be a secondary response to irritation by calculi, lower urinary tract obstruction or in association with renal papillary necrosis as these effects were not observed. Therefore the bladder effects are possibly a primary effect of the test item. The mode of action for this effect is unclear and therefore the effect is used for deriving the NOAEL. There are, however, no indications that the kidney function in females was impaired based on other kidney (related) findings. In addition, there were no related findings seen such as morphological lesions (e.g. inflammation or cell death) and it was not seen in males.

Additionally, an adverse histopathological lesion was recorded in the kidneys of males treated at 2000 ppm. In these males, an increased incidence and severity (up to moderate) of hyaline droplet accumulation was noted. The hyaline droplet accumulation was considered to represent alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia.

This male rat specific protein is not present in female rats nor in higher mammals, including man. In this study the increased hyaline droplet accumulation in males treated at 2000 ppm was accompanied by indicators of renal tubular damage in the form of granular casts at minimal degree and increased tubular basophilia up to slight degree. This combination of findings was considered to be adverse. In addition, males at 2000 ppm had kidney weights that were approximately 15% higher than controls and were considered to be related to the microscopic renal changes.

Higher kidney weights were also recorded for males at 700 ppm (approximately 13% higher than controls), and for females at 250, 700 and 2000 ppm (approximately 13%, 21% and 16% higher, respectively). There were neither corroborative microscopic changes nor clinical biochemical alterations indicative of renal toxicity. Furthermore, kidney weights remained within the range considered normal for this strain of rats of similar age. Therefore, the higher kidney weights for males at 700 ppm and for females at 250 ppm and higher were not considered adverse in nature.

A treatment-related but non-adverse hepatocellular hypertrophy was recorded in the liver of males and females treated at 2000 ppm up to slight degree. This microscopic finding correlated with a modest increase in liver weight (relative weight approximately 13% higher than controls) and macroscopic liver enlargement observed in a few 2000 ppm males. In the absence of any other indicators of hepatocellular toxicity, these liver changes were not considered to be adverse.

No toxicologically relevant changes were noted in clinical biochemistry or haematological parameters up to 2000 ppm.

Under the test conditions, the parental NOAEL in male and female rats was 700 ppm (based on reduced food consumption of females throughout the post-coitum and lactation period at 2000 ppm (approximately 30-40% lower than controls), hypertrophy of the urothelium of the urinary bladder in females at 2000 ppm and renal changes in males at 2000 ppm (hyaline droplet accumulation, accompanied by renal tubular basophilia and granular casts and associated higher renal weight).

NB:

700 ppm in the diet corresponded to mean daily test item intake levels of 51 mg/kg bw/day in males, and 59 (pre-mating period), 87 (post-coitum period) and 129 (lactation period) mg/kg bw/day in females.

2000 ppm in the diet corresponded to mean daily test item intake levels of 150 mg/kg bw/day in males, and 168 (pre-mating period), 184 (post-coitum period) and 280 (lactation period) mg/kg bw/day in females.

Based on the available data on the source substance, the parental NOAEL for the target substance is also set to 700 ppm (the source and the target substance having the same molecular weight, no correction is needed).

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data on the source substance, no additional classification is proposed for the target substance regarding specific target organ toxicity after oral dose-repeated exposure. Indeed, adverse effects were observed at the dose level of 2000 ppm (mean daily test item intake levels of 150 mg/kg bw/day in males, and 168 (pre-mating period), 184 (post-coitum period) and 280

(lactation period) mg/kg bw/day in females), i.e. above the treshold for Category 1 or 2 classification for a study duration > 90 days (10 and 100 mg/kg bw/day, respectively).