3-ethoxy-1,1,5-trimethylcyclohexane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 December 2015 to 17 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 422 without any deviation.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-ethoxy-1,1,5-trimethylcyclohexane

EC Number:

266-722-5

EC Name:

3-ethoxy-1,1,5-trimethylcyclohexane

Cas Number:

67583-77-1

Molecular formula:

C11H22O

IUPAC Name:

3-ethoxy-1,1,5-trimethylcyclohexane

Test material form:

liquid

Details on test material:

- Physical state/Appearance: Clear colorless liquid
- Storage condition of test material: Stored at room temperature in the dark
- Expiration date of the lot/batch: 09 July 2016

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability at higher temperatures: No, maximum temperature: 35°C

OTHER SPECIFICS:
- Volatile: Yes, vapour pressure: 0.6 mmHg (calculated)
- Specific gravity/density: 0.838 g/cm3 at 25 °C

Test animals

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. WIL Research Europe 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 10-12 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: 28 December 2015 to 17 March 2016

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as it is a possible route of human exposure.

Vehicle:

other: feed

Details on oral exposure:

DIET PREPARATION
- 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 the required amount of powder feed. No correction was made for the purity of the test substance.
- Frequency of preparation: Diets were prepared up to 15 days in advance of first use, and were divided into aliquots to be used over 3-4 days at room temperature.
- Storage of preparations: Diets were kept in the freezer (≤-15 °C) until use, if not administered on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 4 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 during the treatment phase, according to a validated method (Test Facility S tudy No. 510235). Samples of diets were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability was determined over 15 days in the freezer (≤ - 15 °C) followed by 4 days at room temperature (highest and lowest concentration). 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%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females were exposed for 42-56 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 ppm

Doses / concentrations 2

Dose / conc.:

5 000 ppm

Doses / concentrations 3

Dose / conc.:

15 000 ppm

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 10-day dose range finding study (Study No. 510232)
- Rationale for animal assignment: 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

Examinations

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.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and twice 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):
- Twice 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 (isoflurane)
- Animals fasted: Yes; animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- Parameters checked:
Haematology: White blood cells (WBC), Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils and 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), Platelets, 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 (Inorg. Phos)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional Observations were performed on the selected 5 animals/sex/group. The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHERS:
General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delive ry 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 in adequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

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.
- Females which delivered: Necropsy was performed on Lactation Days 5-7.
- Females which failed to deliver but with evidence of mating (nos. 53 and 68): Necropsy was performed on post-coitum Day 27 (female no. 68) or Day 34 (female no. 53)*
- Males: Necropsy was performed following completion of the mating period (a minimum of 28 days of dose administration).
* Mating of female no. 53 was overlooked. Reassessment of vaginal smears showed that this animal had mated on 6 February 2016. The necropsy date of this animal (approximately 21 days after the last day of the mating period) was set under the assumption that this animal had not mated.

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, 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 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- Liver and kidneys of all selected males and females of Groups 2 and 3 and thymus and spleen of all selected females, 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 no. 53/male no. 13 and Group 3 female no. 68/male no. 28)
** Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina. All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Statistics:

The following statistical methods were used to analyse 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. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- At 15000 ppm, 6/10 females showed piloerection and one female showed hunched posture on several days during the last week of treatment.
- No clinical signs were noted among females at 1500 and 5000 ppm, and among males of any dose group. No abnormalities were noted during weekly arena observations in any dose group.
CONCLUSION: No adverse effects

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 15000 ppm, mean body weight gain of males was lower than controls throughout the premating and mating period, being statistically significant on most occasions. Slight weight loss (up to 2%) was recorded for 6/10 males over Days 1-4 of the premating period. Mean body weight of males was only statistically significant on Day 4 of the mating period. Overall weight gain of these males was app roximately 6% lower than controls over the complete treatment period.
- Females at 15000 ppm showed a mean body weight loss of up to 5% during the premating and mating period compared to Day 1 of the premating period. Individual weight loss was generally most pronounced over Days 1-4 of the premating period (up to 9% weight loss). During the post-coitum period, mean body weight gain was approximately 15% lower than controls over this period, being statistically significantly on all occasions. During lactation, body weight gain was similar to controls over Days 1-4 of lactation. Mean body weight was approximately 16% lower than controls and was statistically significant on Days 1 and 4 of lactation.
- At 5000 ppm, mean body weight of females was lower than controls throughout the post-coitum and lactation phase, being statistically significant only on Days 1 and 4 of lactation. Weight gain of these females over the post-coitum and lactation period (and during the premating and mating period) remained comparable to controls.
- At 1500 ppm, body weight and body weight gain remained in the same range as controls over the treatment period.
CONCLUSION: No adverse effects

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- At 15000 ppm, a lower food intake before and after correction for body weight was recorded for males and females over Days 1-4 of the premating period. For females, food intake after correction for body weight remained slightly lower than controls during the remainder of the premating period. In the postcoitum and lactation period, food intake before and after correction for body weight remained statistically significantly lower than controls. Food intake (before correction of body weight) of these females was approximately 47% and 48% lower than controls in the post-coitum period (based on mean of means) and lactation period, respectively.
- At 5000 ppm, food intake before and after correction for body weight was statistically significantly lower than controls throughout the post-coitum and lactation period. Food intake (before correction of body weight) of these females was approximately 37% and 30% lower than controls in the post-coitum period (based on mean of means) and lactation period, respectively. Food intake of males at 5000 ppm remained comparable to controls throughout treatment.
- At 1500 ppm, food intake before or after allowance for body weight was similar between treated and control animals.
CONCLUSION: No adverse effects

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

- Haematological parameters of treated rats were not considered to have been affected by treatment.
- Any statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
CONCLUSION: No adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher total protein in males at 5000 and 15000 ppm.
- Higher albumin in males at 5000 and 15000 ppm.
- Higher sodium in males at 5000 and 15000 ppm.
- Lower glucose in males at 5000 and 15000 ppm.
- Lower total bilirubin in males at 15000 ppm.
- Higher creatinine in males at 15000 ppm.
- Higher cholesterol in males and females at 15000 ppm.
- Higher bile acids in females at 15000 ppm.
- Lower alkaline phosphatase activity (ALP) in females at 15000 ppm.
For one female at 15000 ppm (no. 71), a notably lower sodium level was recorded. Since other females at this dose showed a sodium level that was comparable to the control range, this was considered not toxicologically relevant. Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
CONCLUSION: No adverse effects

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 selected animals. Grip strength and motor activity was similar between control and high dose animals. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
- Motor activity (ambulations and total movements) of females at 5000 ppm appeared higher than control means (without achieving a level of statistical significance). Since this variation occurred in the absence of a dose-related trend, this was considered to be of no toxicological significance.
CONCLUSION: No adverse effects

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 15000 ppm treated males (absolute and relative to body weights). Liver to body weight ratio was approximately 24% higher than controls.
- Test item-related higher kidneys weights were noted in the 5000 and 15000 ppm treated males (relative to body weights at 5000 ppm and both absolute and relative to body weights at 15,000 ppm). Kidney to body weight ratio was approximately 22 and 35% higher than controls at 5000 and 15000 ppm, respectively.
- There were no other test item-related organ weight changes. Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of a test item-related effect on final body weight.
CONCLUSION: No adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The following test item-related macroscopic findings were recorded:
- Enlarged liver in 3/10 males at 15000 ppm.
- Enlarged kidneys in 2/10 males at 1500 ppm, 1/10 males at 5000 ppm and 4/10 males at 15000 ppm.
- Pale discolouration of the kidneys in 6/10 males at 15000 ppm.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
CONCLUSION: No adverse effects

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: hepatocellular hypertrophy in males and females at 15000 ppm at minimal degree.
Kidneys:
- Increased incidence and severity of hyaline droplet accumulation in males at 1500, 5000 and 15000 ppm up to marked degree; Increased incidence and severity of tubular basophilia in males at 5000 and 15000 ppm up to moderate severity.
- Tubular granular casts in males at 1500, 5000 and 15000 ppm up to marked degree.
- Tubular degeneration in males at 5000 and 15000 ppm up to slight degree.
- Slightly increased incidence and severity of inflammatory cell infiltrate in males at 15000 ppm.
- Tubular vacuolation in females at 15000 ppm at slight degree.
Thymus: atrophy in females at 5000 and 15000 ppm up to slight degree.
Spleen: decreased incidence and severity of hematopoiesis in females at 5000 and 15000 ppm.
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.
CONCLUSION: No adverse effects

Histopathological findings: neoplastic:

no effects observed

Details on results:

- Reproductive parameters were considered unaffected by treatment.
- Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment. For female nos. 62, 66 and 79, the number of pups was slightly higher than the number of implantations and/or corpora lutea. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 5 of lactation.
- No signs of difficult or prolonged parturition were noted among the pregnant females.
- Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
- No deficiencies in maternal care were observed.
CONCLUSION: No adverse effects

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

729 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

15000 ppm (nominal) = 729 mg/kg bw/day (actual dose received)

2

Key result

Dose descriptor:

NOAEL

Effect level:

725 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

15000 ppm (nominal) = 725 mg/kg bw/day (actual dose received)

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Analysis of Diet Preparations

Accuracy of preparation: The concentrations analysed in the diets of Group 2, Group 3 and Group 4 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 of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability: The diets were not considered stable during storage for 15 days in a freezer (≤ -15°C) in closed container followed by 4 days at room temperature in open container. Relative differences were -29% for Group 2 and -33% for Group 4

 

Table 7.8.1/2: Test article intake

Period	mg substance/kg body weight/day (mean range indicated within brackets)
	Group 2 1500 ppm	Group 3 5000 ppm	Group 4 15,000 ppm
Males
Pre mating	107 (101-111)	359 (346-370)	979 (807-1065)
Mating	109 (96-124)	360 (325-436)	1091 (855-1559)
Mean of means^1	108	360	1041
Females
Pre mating	117 (114-120)	375 (381-382)	937 (1031-1060)
Post-coitum	156 (140-175)	373 (343-414)	1011 (898-1075)
Lactation	271	644	1573
Mean of means^1	152	398	1035

 

1 Mean of means of all periods, weighed for number of measurement intervals per period:

Males: ((4x mean premating) + (5x mean mating)) / 9

Females: ((4 x mean premating) + (6 x mean post-coitum) + (1 x mean lactation)) / 11

 

^.Table 7.8.1/3: Test article intake - corrected

Sex	mg substance/kg body weight/day^1
	Group 2 1500 ppm		Group 3 5000 ppm		Group 4 15,000 ppm
		Corr^2		Corr^2		Corr^2
Males	108	76	360	252	1041	729
Females	152	106	398	279	1035	725

¹Mean of means (weighed for duration of premating, mating, post-coitum and lactation period).

² Corrected for analytical recovery (on approximation30% lower than nominal).

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the parental NOAEL in male and female was 15000 ppm (nominal), corresponding to 729 mg/kg bw/day for males and 725 mg/kg bw/day for females (actual dose received).

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 received test item in the diet, at concentrations of 1500, 5000 and 15000 ppm. Males were exposed for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-56 days, i.e. during 2 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 (twice weekly), clinical pathology (end of treatment), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration and parturition. Diets were analyzed once during the study to assess accuracy, homogeneity and stability.

 

Chemical analysis showed that the diet preparations were prepared accurately and homogenously. Stability analyses indicated that diet preparations were not stable during storage for 15 days in a freezer (≤ -15°C) followed by 4 days at room temperature. Relative differences were -29% at 1500 ppm and -33% at 15000 ppm. The concentrations analysed in the diets at three dose levels were in agreement with target concentrations (i.e. mean accuracies between 80 and 120%).

 

No mortality occurred during the study period. At 15000 ppm, females showed piloerection and hunched posture on several days during the last week of treatment. No clinical signs were noted among females at 1500 and 5000 ppm, and among males of any dose group. No abnormalities were noted during weekly arena observations in any dose group.

 

At 15,000 ppm, slight mean body weight loss / lower weight gain was recorded throughout treatment for males and females, accompanied by a lower food intake during the initial days of treatment. Food intake remained slightly lower for females during the remainder of the treatment period, most notably in the post-coitum and lactation period when food intake was almost half that of control animals. This was accompanied by piloerection shown by most females and hunched posture in one case during the lactation period.

 

At 5000 ppm, food intake was lower for females during the post-coitum and lactation phase, along with a slightly lower mean absolute body weight.

 

At 1500 ppm, body weight and food intake was unaffected by treatment.

 

At 15,000 ppm, higher liver weights were recorded in males, supported by enlargement of the liver in a few males at this dose and a minimal degree of hepatocellular hypertrophy. A minimal degree of hepatocellular hypertrophy was also recorded for females at this dose. This hepatocellular hypertrophy and accompanying liver weight increase occurred in the absence of any other indicators of hepatocellular damage.

 

At 1500 ppm and higher, male kidneys showed increased incidence and severity of hyaline droplet accumulation up to marked degree, which correlated with enlargement, pale discolouration of the kidneys and increased kidney weight in the 5000 and 15,000 ppm males. This increased hyaline droplet accumulation was considered to likely represent alpha2uglobulin, 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. Indicators of tubular damage in this study took the form of granular casts starting at 1500 ppm (up to marked degree) and increased tubular basophilia and tubular degeneration starting at 5000 ppm (up to moderate and slight degree, respectively).

The slightly increased incidence and severity of inflammatory cell infiltrate in the male kidneys at 15,000 ppm likely represents a response to the tubular damage. Alpha2uglobulin is not present in female rats nor in higher mammals, including man. The occurrence of hyaline droplets is a specific male rat response and is not observed in normal female rats, and higher species of either sex, including humans. Therefore, these kidney findings are considered not relevant for human risk assessment.

 

Tubular vacuolation in the kidneys at 15,000 ppm (slight degree) was not considered to be an indicator of tubular damage. A minimal to slight thymus atrophy at 5000 and 15,000 ppm (slight degree) was considered to be secondary to stress, as supported by the reduced body weight. Decreased hematopoiesis in the spleen at 5000 and 15,000 ppm occurred in the absence of haematological changes.

Clinical biochemistry changes at 5000 and 15,000 ppm in both sexes consisted of higher total protein, albumin, sodium, creatinine and cholesterol and lower glucose and total bilirubin in males, and higher bile acids and cholesterol and lower alkaline phosphatase activity in females. At 5000 ppm, changes in clinical biochemistry parameters were confined to higher total protein, albumin and sodium and lower glucose in males. These changes were generally slight in degree (i.e. generally within the range considered normal for rats of this age and strain) and had no apparent histopathological correlates.

 

No treatment-related changes were noted during functional observation tests and haematological assessment.

 

 

Under the test conditions, the parental NOAEL in male and female was 15000 ppm (nominal), corresponding to 729 mg/kg bw/day for males and 725 mg/kg bw/day for females (actual dose received).